Novel isatinyl thiosemicarbazones derivatives as potential molecule to combat HIV-TB co-infection

European Journal of Medicinal Chemistry 46 (20 1 1 ) 106e121

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Novel isatinyl thiosemicarbazones derivatives as potential molecule to combat

HIV-TB co-infection

Debjani Banerjee, Perumal Yogeeswari, Pritesh Bhat, Anisha Thomas, Madala Srividya,

*

Dharmarajan Sriram

Medicinal Chemistry & Antimycobacterial Research Laboratory, Pharmacy Group, Birla Institute of Technology & ScienceePilani, Hyderabad Campus, Jawahar Nagar,

Hyderabad-500 078, Andhra Pradesh, India

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of novel 5-substituted-1-(arylmethyl/alkylmethyl)-1H-indole-2,3-dione-3-(N-hydroxy/methoxy

thiosemicarbazone) analogues were synthesized and evaluated for their anti-HIV activityand anti-tubercular

activity in both log phase and starved cultures. The compound 2-(1-{[4-(4-chlorophenyl)tetrahydropyrazin-1

(2H)-yl]methyl}-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-(methyloxy)hydrazine-1-carbothioa-

Received 16 July 2010

Received in revised form

19 October 2010

Accepted 20 October 2010

Available online 27 October 2010

mide (B21) displayed promising activity against the replication of HIV-1 cells (EC₅₀1.69

bacterial screening B21 proved effective in inhibiting the growth of both log phase (MIC 3.30

(MIC 12.11 M) MTB cultures. Isocitrate lyase enzyme having momentous implication in persistent TB was

mM). In anti-myco-

mM) and starved

m

Keywords:

Isatin

Mannich base

HIV-TB co-infection

Isocitrate lyase

shown tobe inhibited by 1-cyclopropyl-6-ﬂuoro-7-[4-{[5-methyl-3-((Z)-2-{[(methyloxy)amino]carbothioyl}

hydrazono)-2-oxo-1H-indol-1(2H)-yl]methyl}tetrahydropyrazin-1(2H)-yl]-4-oxo-1,4-dihydroquinoline-3-

carboxylic acid (B30) with 63.44% inhibition at 10 mM.

1. Introduction

As stated in a recent review by Pepper et al., dire consequences arise

due to drugedrug and drugedisease interactions and a paradoxical

The acquired immunodeﬁciency syndrome (AIDS) has acquired

pandemic proportion globally and has claimed more lives than any

other diseases. According to the 2008 UNAIDS report released by the

WHO, 33 million people were diagnosed to be human immunode-

ﬁciency virus (HIV)-positive in 2007 [1]. The 2008 WHO report on

tuberculosis (TB), states that 0.7 million cases of HIV-TB co-infection

were reported in 2006 and an estimate of 0.2 million of the global

populace died due to HIV-TB co-infection [2]. TB and HIV have

a harmonized effect on the progression of each other and hence

become a lethal threat to the patient. The therapy available for

treating patients co-infected with HIV and TB involves a very high

pill burden, which reduces the chances of treatment adherence by

the subjects. Current combination therapy for treating patients co-

infected with HIV & TB involves non-nucleoside reverse transcrip-

tase inhibitors (NNRTI) viz. efavirenz, nevirapine, delaviridine and

viral protease inhibitors viz. nelﬁnavir, ritonavir, saquinavir and

a combination regimen comprising of rifampicin (RIF), isoniazid

(INH), ethambutol and pyrazinamide to combat the tubercle bacilli.

aggravation of tuberculous pathologies occur due to reconstitution

of immunity while treating the patients with combination anti-

retroviral and anti-tubercular therapies [3,4]. In patients who have

been exposed to the tubercle bacilli, the chances that the bacilli

persist in a dormant state in the lungs are very high which can lead

to reactivation tuberculosis. This dormant/latent TB evades immu-

nity and also presents itself as an elusive target by the existing anti-

tubercular therapies [5]. Sacchetini et al., in 2000 reported that

isocitrate lyase (ICL), a signiﬁcant enzyme in the glyoxalate shunt

plays a crucial role in the persistence of MTB in the macrophages and

escapes immunity [6].

Isatin derivatives have been long reported for their anti-viral

activities speciﬁcally for theiraction against pox virus [7], vaccinia [8],

rhino virus [9], moloney leukemia virus [10] and sars [11] viruses.

Previous works have also reported the inhibitory activity of isatin-

b-

thiosemicarbazones and isatin derivatives on HIV replication

[12e15]. In this paper, we have reported the synthesis of sixty diverse

N-Mannich base derivatives of 3-(N-hydroxy/methoxy thio-

semicarbazones) of 5-substituted isatins as potential NNRTI which

also inhibited the ICL enzyme of MTB thereby tackling the issues of

dormant tuberculosis, which often presents itself as an opportunistic

infection (OI) in patients afﬂicted with AIDS. Thus we hypothesize

that these new molecules would bypass the pharmacokinetic

* Corresponding author. Tel.: þ91 9010534101; fax: þ91 40 66303998.

E-mail addresses: dsriram@bits-hyderabad.ac.in, drdsriram@yahoo.co m(D. Sriram).

doi:10.1016/j.ejmech.2010.10.020

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

107

interferences of combination HIV-TB therapy, tackle the conse-

3. Biological results and discussion

quences arising due to immune reconstitution and also minimize the

pill burden thereby increasing the prospects of patient compliance.

3.1. In-vitro anti-HIV assays

The titled compounds were tested for their ability to inhibit the

replication of HIV-1 (III_B) cells in MT-4 cells. All the sixty analogues

2. Chemistry

had EC₅₀lying in the range of 1.69

derivatives had EC₅₀less than 10

m

Me129.96

mM (Table 1). Fifteen

The synthesis of the titled compounds of series A (A7eA36) and

series B (B7eB36) was accomplished in four steps and is outlined in

Scheme 1 [16]. When an equimolar amount of N-hydroxylamine

hydrochloride/N-methoxylaminehydrochlorideand carbon disulﬁde

were added to anethanolicsolution ofpotassium hydroxideityielded

the potassium hydroxy/methoxy dithiocarbamates, which when

further reacted with hydrazine hydrate generated the respective

thiosemicarbazide hydrochloride salts as ﬁne needle shaped yellow

crystals. This intermediate was further condensed with various

substituted isatins under the conditions of Schiff reaction in the

presence of sodium acetate, which yielded the corresponding Schiff

bases. The N-Mannich bases were synthesized further by condensing

the acidic imino group of isatin derivatives with formaldehyde and

various secondary amines by irradiating the reaction vessel in

a scientiﬁc microwave reactor for 3e15 min at 455 W. This reaction

was also conducted parallely using conventional technique, but the

reaction took sixty to 72 h to attain completion; whereas when the

reaction was carried out under microwave irradiation, reaction time

was considerably shortened and yield was also enhanced. Moreover

microwave technique is greener since it requires solvent in negligible

amounts and it also obviates the need for work-up.

Elemental and spectral analyses were also performed to assess

the purity of the compounds. IR spectra in general showed an azo-

methine (C]N) peak at 1620 cm^ꢀ1and methylene (Mannich) peak

at 2840 cm^ꢀ1. In the ¹H NMR spectra the signals of the respective

protons of the synthesized compounds were conﬁrmed based on

their chemical shifts, multiplicities and coupling constants. These

spectra showed a singlet at 4.9e5.3 ppm, which corresponds to the

eNCH₂N protons. Elemental analyses results were within ꢁ0.4% of

the theoretical values.

mM. Amongst the thirty hydroxy

thiosemicarbazone derivatives A35 was found to be the most prom-

ising with EC₅₀of 4.18 M. Amongst the various substituted piper-

m

azinyl analogues the most potent ones were found to be the

4-chlorophenyl piperazinyl derivatives (A19eA21) followed by

4-methoxyphenyl piperazine (A22eA24) and thenphenyl piperazine

(A16eA18) derivatives. The presence of dialkylamine at R₁ruled anti-

HIV activity by the following order: N,N-diethylamine> N,N-dime-

thylamine. When R₁was substituted by various ﬂuroquinolones (FQ),

gatiﬂoxacin analogues (A34eA36) showed the greatest inhibition of

HIV replication followed by lomeﬂoxacin derivatives (A31eA33) and

then by ciproﬂoxacin (A28eA30) and norﬂoxacin analogues

(A25eA27) which were almost equipotent to each other. The

methoxy thiosemicarbazone analogues when compared to those of

hydroxy thiosemicarbazone derivatives displayed better inhibitory

proﬁle. The higher activity of the methoxy thiosemicarbazones in

comparison to the hydroxy thiosemicarbazones can be attributed to

the methoxy moiety, being involved in steric interaction with the

aromaticresiduesofHIV-1RT;whichwasconﬁrmedwiththedocking

results obtained. In the series of methoxy thiosemicarbazone

analogues, 2-(1-{[4-(4-chlorophenyl) tetrahydropyrazin-1(2H)-yl]

methyl}-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-(meth-

yloxy)hydrazine-1-carbothioamide (B21) was found to be the most

potent in suppressing the replication of HIV-1 cells with an EC₅₀of

1.69 mM. The anti-HIV potency of B21 may be ascribed to the elec-

tronegative chlorine present at the 4th position of phenyl piperazinyl

methyl which probably forms an lipophilic interaction in the active

site pocket and also owing to the methyl group at the 5th position of

the indolyl moiety which helps it in establishing lipophilic

H N

H

N

NH₂.HCl

N

SK

ii

i

C

R

O

C

.HCl

R

O

NH₂

R

O

S

(A3) R=H

(B3) R=CH₃

(A1) R=H

(B1) R=CH₃

(A2) R=H

(B2) R=CH₃

H

N

H

S

C

S

N

C

R

O

NH

N

NH

N

iii

iv

R'

O

N

H

H₂C

R₁

(A4) R=H, R'=Cl

(A5) R=H, R'= F

(A7-A36) R=H

(B7-B36) R=CH₃

(A6) R=H, R'=CH₃

(B4) R=CH₃, R'=Cl

(B5) R= CH₃,R'= F

(B6) R=CH₃, R'= CH₃

Scheme 1. Synthetic protocol of compounds. Reagents & Conditions: (i) CS₂, KOH, C₂H₅OH, 0e5 ^ꢂC, 0.5 h; (ii) NH₂NH₂.H₂O, 80 ^ꢂC, 1 h, conc. HCl; (iii) 5-R⁰-Isatin (R⁰¼ Cl, F, CH₃),

CH₃COONa; (iv) 30% HCHO, NHR₁, M.W.I, 455 W, 3e15 min.

108

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

Table 1

Physical Constants, anti-HIV activity and anti-tubercular activity of A7e36, B7e36.

Sl. No.

R⁰

R₁

M.P(^ꢂC)

% yield

HIV-1

MTB MIC^c

(mM)

a

b

EC₅₀

(

mM)

CC₅₀(mM)

A7

B7

A8

B8

A9

B9

A10

B10

A11

B11

A12

B12

Cl

F

CH₃

Cl

F

—N(CH₃)₂

-do-

—N(C₂H₅)₂

-do-

102e104

60e62

40e42

56e58

110

82e84

125e127

52e54

80e82

98e100

114e116

88e90

97.05

98.28

87.10

82.45

95.85

93.45

98.61

69.28

79.90

83.50

71.47

68.81

129.96

57.93

47.22

37.80

113.22

64.09

120.84

58.40

36.24

29.71

57.61

53.27

610.15

551.75

599.15

559.69

650.68

622.28

562.05

454.21

520.93

490.07

534.25

572.80

76.27

18.28

10.05

9.62

81.34

19.45

70.26

16.90

9.22

F

CH₃

4.41

11.83

8.96

-do-

A13

Cl

46e48

56.43

35.13

347.72

6.11

B13

A14

B14

A15

B15

Cl

F

CH₃

-do-

72e74

36e38

80e84

58e62

86e90

98.90

64.75

89.53

64.47

75.70

28.40

61.41

28.85

93.59

45.13

322.26

465.01

274.09

572.39

365.41

4.06

4.41

4.25

8.96

4.29

A16

Cl

148e150

87.21

28.32

273.07

28.09

B16

A17

B17

A18

B18

Cl

F

CH₃

-do-

80e82

78e80

108e110

68e70

93.33

86.09

79.94

79.93

83.57

17.65

19.60

18.08

19.32

17.3

267.13

250.89

226.89

420.00

276

13.62

7.30

7.07

14.72

12.36

87e89

A19

Cl

110e112

92.40

11.06

268.14

13.04

B19

A20

B20

A21

B21

Cl

F

CH₃

-do-

208e210

72e74

202

178e182

140

74.26

73.50

76.87

76.34

85.46

2.43

6.85

3.77

6.10

1.69

228.61

193.55

346.79

335.11

300.22

6.34

3.37

3.27

6.82

3.30

A22

Cl

140

58.32

21.26

278.76

13.16

B22

A23

B23

A24

B24

Cl

F

CH₃

-do-

108e110

54e56

116e118

216e218

98e102

73.92

60.38

69.35

88.66

61.11

13.09

14.66

12.7

14.96

9.82

220.86

259.54

276

320.98

324.39

12.78

6.83

6.14

13.75

6.68

A25

Cl

166e168

71.55

27.24

185.54

1.30

B25

A26

B26

A27

Cl

F

-do-

170e174

88e90

66e68

98.59

72.88

93.50

47.33

19.15

18.44

10.51

16.88

192.51

207.66

143.76

326.67

0.65

1.33

0.67

1.34

CH₃

180e182(d)

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

109

Table 1 (continued ).

Sl. No.

R⁰

R₁

M.P(^ꢂC)

% yield

67.23

HIV-1

MTB MIC^c

1.31

(mM)

a

b

EC₅₀

(

m

M)

CC₅₀(mM)

B27

A28

CH₃

-do-

158e162(d)

11.42

237.72

Cl

210(d)

92.05

33.38

229.30

2.54

B28

A29

B29

A30

B30

Cl

F

CH₃

-do-

128e130(d)

230(d)

184(d)

72.53

90.62

65.08

71.67

85.67

10.99

16.50

11.43

20.89

9.22

142.66

222.23

254.78

303.72

231.71

0.64

0.33

0.31

0.67

0.66

40

150e154(d)

A31

Cl

226e228

88.59

23.18

209.45

1.23

B31

A32

B32

A33

B33

Cl

F

CH₃

-do-

>230

130(d)

180(d)

140e144

206e210(d)

48.33

61.75

72.90

72.36

81.22

9.26

9.93

4.43

11.73

5.74

180.38

186.85

178.27

243.95

155.50

1.20

0.65

0.63

0.65

1.24

A34

Cl

136e138

90.92

15.35

233.73

1.19

B34

A35

B35

A36

B36

Nevirapine

Troviridine

Cl

F

CH₃

e

-do-

e

154e156

70e74

88e90

70

70e72

e

8.47

66.79

70.23

77.75

46.09

e

7.14

4.18

1.88

11.13

1.86

0.13

164.25

205.88

170.51

276.00

154.06

156

0.30

0.16

0.15

0.63

0.31

e

0.016

87

e

a

Effective concentration of the drug that reduces HIV-1 induced cytopathic effect by 50% in MT-4 cells.

Cytotoxic concentration of the drug that decreases MT-4 cell’s viability by 50%.

MIC was the concentration at which the H₃₇Ra MTB strain showed complete inhibition.

b

c

interactions with aromatic residues of the enzyme. Between mor-

contributingtoimprovedinteractionwiththebulkygroupspresentat

the active site. Substitution at R₁also ruled the pattern of activity

followed by the compounds. Presence of piperazinyl methyl and

substituted piperazinylmethyl group at N-1 enhanced the anti-HIV

activity when compared to morpholinylmethyl group. This can be

attributed to the repulsion experienced by the oxygen of morpholine

ring.

The cytotoxic effect of these molecules was measured in MT-4

cells in parallel with the anti-viral activity. Amongst the entire series

A9 was found to be least cytopathic to MT-4 cells with a CC₅₀value of

pholinyl and piperazinyl substitution at R₁of the methoxy thio-

semicarbazones, piperazinyl analogues (B16eB24) showed improved

anti-HIV potency. Comparison between the various substituted

piperazinyl showed that 4-chlorophenyl piperazine derivatives

(B19eB21) were most active followed by 4-methoxyphenyl pipera-

zine (B22eB24) and lastly phenyl piperazinyl analogues (B16eB18).

4-Chlorophenyl piperazinyl methyl substituted molecules showed

higher inhibitory activity against replication of HIV-1 cells in

comparison to other phenyl piperazines namely phenyl piperazine

and 4-methoxyphenyl piperazine due tothehalogen group presentat

the 4th position of the phenyl ring forming a bond with some posi-

tivelychargedresiduesoftheRTenzyme. PresenceofvariousFQsatR₁

650.68

m

M. The CC₅₀values of the entire series of sixty compounds

M (Table 1). When

were found to lie in the range of 143.76e650.68

m

compared to standard non-nucleoside reverse transcriptase inhib-

itors (NNRTI) like nevirapine and delaviridine, which have CC₅₀of

guides

activity

in

the

following

sequence:

gatiﬂoxacin > lomeﬂoxacin > ciproﬂoxacin z norﬂoxacin. The

presence of FQs at N-1 makes the molecule B36 the second most

potent molecule amongst the total 60 reported compounds against

HIV replication. When considering the substituent at 5th position of

the indole-2,3-dione it was observed that activity was governed by

the order of F z CH₃> Cl. Compounds with N, N-diethylaminomethyl

substitution at N-1 of indole-2,3-dione (A10eB12) showed better

inhibitory activity than those with N,N-dimethylamine methyl

substitution(A7eB9)possiblydue tothelongeraliphaticchainlength

around 25 mM, these new molecules are far less cytotoxic. Cyto-

toxicity was found to be regulated by the occurrence of greater bulky

groups at N-1 of indolyl moiety. While comparing between the

terminal substituent present at the hydrazino carbothioamide it was

seen that molecules belonging to series of methoxy thio-

semicarbazone analogues were mostly less toxic than that of the

series of hydroxythiosemicabazones except for a few isolated cases,

which can be ascribed to the probable hydrogen bond formation by

the hydroxyl group with residues of the MT-4 cells.

110

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

3.2. HIV-1 RT enzyme inhibition assay

ﬂuorine > chlorine z methyl, with moderate to lowest activity

swaying between chlorine and methyl, which might be due in part to

the combined effect of the substituent present at R₁.

HIV-1 reverse transcriptase (RT) is a predominant target for many

anti-HIV drugs that are being used clinically and are there in the

developmental stages. RT is a multifunctional heterodimeric enzyme

needed in HIV replicative cycle, which catalyzes the conversion of

genomic HIV RNA into proviral DNA [17,18]. Drugs targeting RT are

classiﬁed into two broad categories: nucleoside reverse transcriptase

inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibi-

tors (NNRTI). NNRTIs inhibit RT in a non-competitive manner by

binding at an allosteric site that is situated 10 Å away from the

catalytic HIV-RT site [19]. NNRTIs are speciﬁc to HIV-1 RT and hence

are comparatively less toxic to the host. Though NNRTIs are struc-

turally diverse, they act on RT by inducing conformational changes

that form the NNRTI binding pocket (NNIBP). They achieve this by

distorting the polymerase active site by means of movement of the

catalytic aspartates [20].

Compound B35 was found to be the most promising in the

methoxy thiosemicarbazone series. Varying substituents at R₁led

to the same order of activity as that of hydroxy thiosemicarbazones.

The greater activity proﬁle of the ﬂouroquinolone-substituted

analogues (A25eB36) can be accredited to their increased lip-

ophilicity permitting them to penetrate into the mycolic acid cell

wall of the bacilli. In case of the morpholinyl derivatives (A13eB15)

the enhanced activity in contrast to the piperazinyl derivatives may

be due in part to the electronegative oxygen which might be

involved in some ionic interaction or some hydrogen bonding

interaction with residues at the active site. Amongst the substituted

piperazinyl analogues, the 4-chlorophenyl piperazinyl analogues

demonstrated highest activity, which can be attributed to the

chlorine atom being involved in some probable ionedipole inter-

action with the residues of the active site pocket. In the presence of

dialkylamino group at R₁, increased alkyl chain length contributed

to greater inhibition of MTB replication possibly due to increased

lipophilic factor. These results were further established by the

docking simulations that were performed.

Four compounds was assessed for their activity against wild

type HIV-1 RT. Compound B21 showed the highest inhibitory

activity with IC₅₀of 11.5 ꢁ 1.5

mM (Table 2) on HIV-1 RT amongst all

four, which can be ascribed to the methyl moiety present at thio-

semicarbazo moiety, which may be involved in lipophilic interac-

tion with some residues present in the RT active site. Activity can

moreover be guided by the phenyl ring attached to the piperazinyl

moiety at R₁, which can contribute to signiﬁcant interaction with

aromatic residues of the RT enzyme as is evident from the docking

simulations performed.

These compounds also showed exemplary inhibitory proﬁle

against starved MTB culture. Twenty compounds from amongst the

sixty derivatives were tested for their activity against the replication

of dormant MTB culture (Table 3). Compound B35 proved superior

in curbing the growth of dormant MTB (MIC: 9.17

to ﬁrst-line anti-tubercular drugs, INH (MIC: 182.31

(MIC: 15.19 M). All the twenty analogues were found to be more

active (MIC ranging from 9.17 to 42.11 M) in suppressing the

m

M) in comparison

m

M) and RIF

m

3.3. In-vitro anti-tubercular evaluation

m

proliferation of dormant MTB when compared to INH. Seven of them

were found to be more beneﬁcial (MIC ranging from 9.17 to

13.48 mM) than RIF in inhibiting dormant MTB.

The titled compounds when evaluated for their inhibitory activity

against the replication of MTB in the logarithmic growth phase dis-

played activity ranging from 0.15 to 81.34

compounds had minimum inhibitory concentrations (MIC) below

10 M out of which ﬁfteen molecules’ MIC were found to lie below

M. 1-Cyclopropyl-6-ﬂuoro-7-[4-{[5-ﬂuoro-3-((Z)-2-{[(methyloxy)

mM (Table 1). Forty-four

m

3.4. MTB ICL enzyme inhibition assay

1

m

amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H)-yl]methyl}-3-

methyltetrahydropyrazin-1(2H)-yl]-8-(methyloxy)-4-oxo-1,4-dihy-

droquinoline-3-carboxylic acid (B35) was found to be the most active

In chronic TB, the bacillus exists in diverse metabolic phases,

spanning from active cell growth to stationary phase [13]. All the

currently available anti-tubercular drugs that are being clinically

againstthelogarithmicgrowthofMTBwithMICof0.15

mM, even more

potent than INH (MIC: 0.36 M). In contrast to the hydroxy thio-

m

Table 3

semicarbazone analogues, the methoxy thiosemicarbazones exhibi-

ted superior activity against MTB growth. Amongst the hydroxy

thiosemicarbazone analogues the most active compound was found

Comparison between anti-tubercular activity of selected compounds against log

phase and dormant MTB.

to be A35 with an MIC of 0.16 m

M (Table 1). Keeping R⁰constant in the

Compd

MTB in log phase

M)

Dormant MTB

(mM)

(

m

hydroxysemicarbazone series when R₁wasvaried the orderof activity

was as follows: N,N-dimethylamino < N,N-diethylamino < phenyl

A20

A21

A29

A30

A32

A33

A35

A36

B14

B19

B20

B21

B24

B25

B26

B28

3.37

6.82

0.33

0.67

0.65

0.16

0.63

4.25

6.34

3.27

3.30

0.30

0.65

0.67

0.64

0.66

0.63

0.15

0.31

0.36

0.12

13.48

27.28

20.92

42.11

10.12

20.37

9.74

19.60

17.01

25.36

13.08

12.11

38.01

36.86

19.24

34.62

18.44

9.45

piperazinyl

< 4-methoxyphenyl piperazinyl < 4-chlorophenyl

piperazinyl < morpholinyl < norﬂoxacin

< ciproﬂoxacin < lomeﬂoxacin < gatiﬂoxacin. Substituents at R⁰

inﬂuenced the anti-tubercular activity in the following order:

Table 2

HIV-1 RT enzyme Inhibition Data for Substituted Isatin-

b-

thiosemicarbazones^a.

Compd

IC₅₀(m

M) against HIV-1 RT^b

A21

B19

B20

B21

Nevirapine

Troviridine

23.4 ꢁ 1.2

18.3 ꢁ 2.1

16.7 ꢁ 2.3

11.5 ꢁ 1.5

B30

B32

B35

B36

Isoniazid

Rifampicin

0.25

9.17

17.81

182.31

15.19

0.017 ꢁ 0.007

a

Enzyme assays done with WT RT.

IC₅₀is the quantity of drug that reduced WT RT enzyme activity by

b

50%.

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

111

used do not target various stages that MTB uses for its persistence

in the macrophages and hence prolonging the treatment regimen.

A lot of research activity is currently aimed at understanding the

biology of persistence of the tubercle bacillus and developing new

drugs that target the persister bacteria [21]. Among the gene

products involved in mycobacterial persistence, isocitrate lyase

(ICL) [22] could be good targets for the development of drugs that

target persistent bacilli. As the compounds reported in this article,

demonstrated excellent activity against dormant mycobacterium,

we decided to explore the possible mechanism by screening some

compounds against ICL enzyme of MTB. Several small-molecule

inhibitors have been described [23] as MTB ICL inhibitors; however,

none has been developed as a drug for MTB. In this work, isocitrate

lyase activity was determined at 37 ^ꢂC by measuring the formation

of glyoxylate-phenylhydrazone in the presence of phenylhydrazine

and isocitrate lyase at 324 nm based on the method described [24].

The compounds were screened with a single concentration of

produced the highest q²of 0.666 at 6 components with high

conventional r². The obtained model was statistically signiﬁcant as

was evident from the q², s_press, SE, F-value and r²_bsvalues tabulated

in the table for CoMFA and Advanced CoMFA ﬁelds. This ﬁnal

CoMFA model, had a cross-validated r²(q²) of 0.666 with 6 principal

components. The non cross-validated r²was determined to be

0.955 with standard error of estimate (SE) being 0.108 and covari-

ance ratio (F) being 99.186 (signiﬁcant at 99% level). This model was

further validated using the test set compounds, which conﬁrmed

its reliable predictive ability. Bootstrapping results also supported

the reliability of this model. The statistical parameters of this model

are listed in Table 5.

In the CoMFA model, the steric and electrostatic ﬁeld contribu-

tions were 0.669 and 0.331 respectively. The steric and electrostatic

contour maps of this model are shown in Fig. 1A and B respectively.

The red regions in the contour maps refer to the electrostatically

favored region and the blue region corresponds to electrostatically

un-favored region. The green polyhedric regions correspond to

sterically favored regions and yellow region refer to the sterically

un-favored region. The red region near the para position of the

phenyl ring of the phenyl piperazinyl methyl moiety suggests that

introduction of more electronegative group at this position would

enhance the anti-HIV activity of this class of compounds by virtue of

some strong electrostatic interactions. The blue regions near the 5th

and 6th carbons of the phenyl ring indicate that presence of any

electronegative moiety at these positions would be detrimental for

anti-viral activity. The blue polyhedra around C₇of isatin and C₂of

piperazine also indicated that introduction of any electronegative

group at these positions would diminish the potential of these

compounds to inhibit HIV. In the steric contour maps the green

contour zone around para position of phenyl ring depicts that

bulkier groups would enhance the anti-HIV activity. The diminutive

green polyhedra near the methoxy group attached to the thiourea

revealed that introduction of moderate bulky groups would be

beneﬁcial for anti-HIV activity. The yellow region near the methyl

group at C₅of isatin demonstrates that presence of bulky substitu-

ents would negatively inﬂuence the anti-HIV proﬁle.

10

with the standard MTB ICL inhibitor 3-nitropropionic acid (3-NP)

(at 100 M) for comparison are reported (Table 4).

mM and percentage inhibitions of the screened compounds along

m

Among nine compounds which were assessed for their ability to

inhibit ICL of MTB, B30 demonstrated excellent inhibitory action of

the ICL enzyme of about 63.44% and was more potent than standard

ICL inhibitors like 3-NP. Compounds B20 and B24 showed mild ICL

inhibition with range of 20e30%. Amongst the hydroxy thio-

semicarbazone analogues evaluated, A20 showed an inhibition of

18.54%. The inhibitoryactivities of B24 and B30 can be assigned to the

methyl group present at R⁰and also to the bulky groups present at R₁,

which might be contributing to signiﬁcant lipophilic interactionwith

bulky residues present in the enzyme active pocket. This was also

conﬁrmed by the docking study performed.

4. Molecular modeling analysis

4.1. CoMFA and CoMSIA analyses based on HIV-1 screening

The CoMFA analysis was performed by dividing the array of

compounds into training set of 34 compounds for model develop-

ment and test set of 20 analogues, which were used for validation of

this model. CoMFA study was developed using steric and electro-

static ﬁelds as independent variable and pEC₅₀as dependent vari-

able. CoMSIA study was developed based on steric, electrostatic,

hydrophobic, hydrogen bond donor and hydrogen bond acceptor

similarity ﬁelds as independent variables and pEC₅₀as dependent

variable.

The best CoMSIA model, with q²of 0.823 is the one when all

steric, electrostatic, hydrophobic, acceptor and donor ﬁelds were

taken into account, and the contributions by these ﬁelds were

0.123, 0.241, 0.329, 0.242 and 0.064 respectively (Table 5). The

donor ﬁeld being comparatively insigniﬁcant was ignored for

further calculations. The CoMSIA hydrogen bond acceptor contour

illustrates (Fig. 2B) that presence of hydrogen bond acceptor groups

(magenta colored) at the position adjacent to the thiourea moiety,

The ﬁnal CoMFA model was generated using cross-validated PLS

analysis. PLS analysis showed a high q²value of 0.642 at 6

components with parabolic ﬁeld (advanced CoMFA). The other

ﬁelds like the indicator ﬁeld and H-bonding ﬁeld showed q²of

0.586 and 0.356 at 4 and 6 components respectively. When all these

regions were focused, the q²improved and the parabolic ﬁeld

Table 5

Summary of CoMFA, Advanced CoMFA and CoMSIA results.

Parameters

CoMFA

SCT(RF)

CoMSIA

SEADH

PBL

0.642

0.290

0.971

0.086

156.397

6

PBL(RF)

q²

0.71

0.262

0.970

0.089

148.08

6

0.666

0.280

0.955

0.108

99.186

6

0.823

0.209

0.970

0.088

151.306

9

Table 4

s_press

MTB ICL suppression by selected compounds.

r²

SE

F

N

fractions

S

E

Compound code

MTB ICl % Inhibition

A20

A21

B12

B14

B19

B20

B21

B24

B30

3-NPA

18.54 (10 mM)

11.03 (10 mM)

18.9 (10 mM)

15.93 (10 mM)

6.38 (10 mM)

28.37 (10 mM)

9.49 (10 mM)

23.3 (10 mM)

63.44 (10 mM)

65.99 (100 mM)

0.653

0.347

e

0.669

0.331

e

0.954

0.046

e

0.123

0.241

0.329

0.242

0.064

0.981

0.005

0.792

H

A

D

e

r²

0.984

0.007

0.694

0.986

0.003

0.661

0.978

0.007

0.632

bs

SD_bs

r²

cv

112

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

Fig. 1. A: CoMFA contour map of steric regions (green, favored; yellow; disfavored) around B21; B: CoMFA contour map of electrostatic regions around B21. Blue regions are favored

by positively charged groups and red regions favored by negatively charged groups. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the

web version of this article).

around the piperazine moiety and the phenyl ring attached to the

piperazine ring are conducive for the anti-retroviral activity of this

series. The CoMSIA hydrophobic contour map (Fig. 2A) depicted

that presence of more hydrophobic moieties around the phenyl

ring of isatin, piperazine and surrounding the phenyl appended to

the piperazinyl methyl groups would prove to increase anti-HIV

activity. The yellow polyhedron, in the CoMSIA steric contour,

(Fig. 2C) near the chlorine attached to the para position of phenyl

ring and C₅of phenyl ring demonstrates that presence of bulky

groups at these positions would hinder anti-viral activity. The green

region around C₁, C₂and C₆of phenyl ring emphasizes that bulky

groups at these positions would further enhance the anti-HIV

activity. The CoMSIA electrostatic contour map (Fig. 2D) demon-

strated that presence of electronegative group at para position of

the phenyl ring would further increase the anti-retroviral activity as

evident by the red region.

4.2. Docking simulation

4.2.1. HIV-1 RT docking

In order to probe the binding mode of our newly synthesized

compounds in the HIV-RT active site, a modeling study was per-

formed by means of GOLD for docking. Compound B21 was docked

onto the active site pocket of HIV-1 RT, the coordinates of which

were taken from the crystal structure of RT/Nevirapine complex. The

docking view of B21 (Fig. 3A and C), exhibited that the NH moiety of

the methoxythioamido group was engaged in a hydrogen bonding

interaction with the ring nitrogen of Pro 140. The nitrogen of the

terminal N-methoxy group was hydrogen bonded to the alcoholic

oxygen of Tyr 181. The oxygen atom of the methoxy group was

hydrogen bonded to the nitrogen of Gln 182⁰s amide. The oxygen at

C₂of the indole ring was involved in hydrogen bond formation with

the backbone nitrogen of Gln 91. The thioamido sulfur formed

Fig. 2. A: CoMSIA contour map of hydrophobic regions (yellow favored; white disfavored) around B21; B: CoMSIA hydrogen bonding acceptor contour map (magenta favored; green

disfavored) around B21; C: CoMSIA steric contour map (green favored; yellow disfavored) around B21; D: CoMSIA electrostatic contour map around (red favored; blue electro-

negative) B21. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

113

Fig. 3. A: Contact residues of B21 when docked onto HIV-1 RT; B: Contact residues of B30 when docked onto MTB ICL; C: Electrostatic surface view of active site pocket of HIV-1 RT

bound to B21; D: Electrostatic surface view of active site pocket of MTB ICL bound to B30.

a hydrogen bond with Tyr 181. The chlorine functional group at the

para position of the phenyl ring of the phenyl piperazinyl methyl

group was involved in electrophilic interactionwith Lys 166. Pro 140

was the major contributor of hydrophobic interaction for the

aromatic indole nucleus. The aliphatic chains of Glu 89 and Gln 91

also interacted hydrophobically with the indole moiety. The

aromatic ring of Tyr 181 and the aliphatic chain of Gln 182 were

involved in steric interaction with the terminal methoxy group. The

chlorophenyl ring was well accommodated in the pocket bordered

by the hydrophobic amino acid residues Trp 88, Val 90 and Lys 166.

These residues also form the binding pocket for standard NNRTIs

[25]. In summary, the results of the GOLD docking results supported

our designed and synthesized compounds. Further structural opti-

mization of B21 for enhanced anti-HIV activity will be based on

these facts highlighted by this docking simulation.

the active site pocket, which was deﬁned by a distance of 11.0 Ǻ from

the centre of the active site. The docking analysis afforded a view

(Fig. 3B and D) of the hydrogen bonding interactions between the

ligand and the active site residues of the protein. The hydroxyl of the

quinolinyl carboxylate was engaged in hydrogen bonding interac-

tions with Tyr 89, Ser 91, Gly 92 and Trp 93. The carbonyl oxygen of

the quinolinyl carboxylate was hydrogen bonded to Tyr 89. The

carbonyl oxygen at C₄of quinoline moiety was involved in hydrogen

bonding with the nitrogen of the peptide backbone of Trp 93. The N₄

of piperazine was found to be hydrogen bonded to imidazolyl

nitrogen of His 193. The ﬂuoroquinoline moiety was nestled in the

hydrophobic pocket formed by the aromatic Trp 93, Leu 194, Thr 347

and Leu 348. Speciﬁcally, Trp 93 by virtue of its

p-cloud provided

favorable aromatic interaction with the quinolinyl moiety. The

cyclopropyl moiety attached to the N-1 of quinoline was located in

the pocket formed by Trp 283, Pro 316, Thr 347 and Leu 348. The

ﬂuorine atom at C₆of quinoline was hydrogen bonded to Ser191 and

it also possessed electrophilic interactions with Trp 93, Leu 194 and

Leu 348. The carboxylate moiety at C₃was also hydrogen bonded to

the metal atom, Mg^þþion of the protein. These residues also have

signiﬁcant implications in binding prototype ICL inhibitors viz. 3-NP

and 3-bromo pyruvate [6].

4.2.2. MTB ICL docking

With the intention of investigating the binding manner of our

designed analogues, a docking simulation was performed using

Autodock 4.0. Compound B30 was docked into the active site pocket

of MTB ICL, the coordinates of which were taken from the crystal

structure of ICL/Pyruvate complex. Eleven residues were present in

114

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

5. Conclusion

stretch), 3300 (amide NH stretch); ¹H NMR (DMSO-d₆, 400 MHz)

7.02 (d, 1H at C₆of indolyl), 7.37 (d, 1H at C₇of indolyl), 7.61 (s, 1H

d

B21 was found to be the most potent in inhibiting the replica-

tion of HIV-1 cells. It also inhibited HIV-1 RT to moderate extent

and even had moderate inhibitory proﬁle against MTB ICL. By

taking into account the facts highlighted by the CoMFA-COMSIA

studies and the docking analyses B21 can be further modiﬁed

structurally for optimal activity against both HIV and TB infection;

thereby proving efﬁcacious against HIV-TB co-infection.

at N1 of indolyl), 7.79 (s, 1H at C₄of indolyl), 8.52 (s, 1H, hydrazino),

9.47 (d, 1H, thioamido), 10.51 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆,

400 MHz) d 111.74 (C₆of indolyl), 115.22 (C₄of indolyl), 116.74 (C₇of

indolyl), 125.51 (C_4aof indolyl), 131.03 (C₃of indolyl), 138.1 (C_7aof

indolyl), 151.53 (C₅of indolyl), 152.48 (C of carbothioamido), 154.06

(C₂of indolyl); Anal[calculated/found]. C, 42.52/42.56; H, 2.78/2.72;

N, 22.04/22.08C₉H₇FN₄O₂S.

6. Experimental

6.1.2.3. (3Z)-5-Methyl-1H-indole-2,3-dione 3-(N-hydroxy thiosemi-

carbazone) (A6). %Yield: 63.63; M.P: 197e200 ^ꢂC; ¹H NMR (DMSO-

6.1. Chemistry

d₆, 400 MHz) d 2.31 (s, 3H, CH₃at C5 of indolyl), 6.75 (d, 1H at C₆of

indolyl), 7.03 (d, 1H at C₇of indolyl), 7.43 (s, 1H at C₄of indolyl), 7.57

(s, 1H at N₁of indolyl), 8.51 (s, 1H, hydrazino), 9.48 (d, 1H, thio-

amido), 10.52 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

Melting points were determined on an electro thermal melting

point apparatus (Buchi BM530) in open capillary tubes and are

uncorrected. All the synthetic steps were monitored till comple-

tion using thin layer chromatography. IR spectra for the

compounds were recorded using Jasco IR Report 100 (KBr pellet)

Spectrophotometer. ¹H NMR spectra were recorded on a JEOL Fx

400 MHz NMR spectrometer using DMSO-d₆as solvent. Chemical

d

20.58 (CH₃), 113.37 (C₇of indolyl), 126.05 (C_4aof indolyl), 127.6 (C₆

of indolyl),129.33 (C₅of indolyl),129.78 (C₄of indolyl), 133.13 (C₃of

indolyl), 140 (C_7aof indolyl), 152.48 (C of carbothioamido), 154.06

(C₂of indolyl); Anal[calculated/found]. C, 47.99/47.95; H, 4.03/4.07;

N, 22.39/22.35; C₁₀H₁₀N₄O₂S.

shifts are expressed in

d (ppm) relative to tetramethylsilane.

Elemental analyses (C, H, and N) were performed on Perkin Elmer

model 240C analyzer and the data were within ꢁ0.4% of the

theoretical values.

6.1.2.4. (3Z)-5-Chloro-1H-indole-2,3-dione 3-(N-methoxy thiosemi-

carbazone) B4. %Yield: 35.63; M.P: 223e225 ^ꢂC; ¹H NMR (DMSO-

d₆, 400 MHz) d 9.2 (s, 1H, thioamido), 8.9 (s, 1H, hydrazino), 7.92 (s,

1H at C₄of indolyl), 7.78 (d, 1H at C₆of indolyl), 7.61 (s, 1H at N₁of

indolyl),7.43 (d, 1H at C₇of indolyl), 3.41 (s, 3H, CH₃of methoxy);

6.1.1. General method for the synthesis of thiosemicarbazides (A3, B3)

To a solution of N-hydroxylamine(A1)/N-methoxylamine(B1)

(0.01 mol) in absolute ethanol (20 mL) was added potassium

hydroxide (0.01 mol) and carbon disulphide (0.75 mL), and the

mixture was stirred at 0e5 ^ꢂC for 1 h to form the corresponding

potassium salt of dithiocarbamates (A2,B2). To the stirred mixture

of dithiocarbamate salts was added hydrazine hydrate (0.01 mol)

and the stirring was continued at 80 ^ꢂC for 1 h and on adding upon

crushed ice the corresponding thiosemicarbazide was obtained

which is converted to hydrochloride salts (A3,B3). A3: Yield: 6.96%;

M.P: 258e260 ^ꢂC Anal[calculated/found]. C, 11.21/11.19; H, 4.70/

4.68; N, 39.22/39.10 CH₅N₃OS; B3: Yield: 50.96%; M.P: 100e102 ^ꢂC

Anal[calculated/found]. C, 19.83/19.80; H, 5.82/5.87; N, 34.68/

34.61C₂H₇N₃OS.

¹³C NMR (DMSO-d₆, 400 MHz)

d 65.37 (C of methoxy thio-

semicarbazone), 117.17 (C₇of indolyl), 118.7 (C_4aof indolyl), 126.97

(C₄of indolyl), 127.36 (C₅of indolyl), 132.14 (C₆of indolyl), 133.18

(C₃of indolyl), 139.86 (C_7aof indolyl), 154.06 (C₂of indolyl), 158.93

(C of carbothioamido); Anal[calculated/found]. C, 42.18/42.22; H,

3.19/3.23; N, 19.68/19.65C₁₀H₉ClN₄O₂S.

6.1.2.5. (3Z)-5-Fluoro-1H-indole-2,3-dione 3-(N-methoxy thiosemi-

carbazone) B5. %Yield: 27.54; M.P: 214e216 ^ꢂC; ¹H NMR (DMSO-d₆,

400 MHz)

d 9.25 (s, 1H, thioamido), 8.94 (s, 1H, hydrazino), 7.91(s,

1H at C₄of indolyl), 7.81 (d, 1H at C₆of indolyl), 7.63 (s, 1H at N₁of

indolyl), 7.46 (d, 1H at C₇of indolyl), 3.45 (s, 3H, CH₃of methoxy);

¹³C NMR (DMSO-d₆, 400 MHz)

d 65.37 (C of methoxy thio-

semicarbazone), 111.74 (C₆of indolyl), 115.22 (C₄of indolyl), 116.74

(C₇of indolyl), 125.59 (C_4aof indolyl), 131.03 (C₃of indolyl), 138.1

(C_7aof indolyl), 151.53 (C₅of indolyl), 154.06 (C₂of indolyl), 158.93

(C of carbothioamido); Anal[calculated/found]. C, 44.77/44.59; H,

3.38/3.36; N, 20.88/20.96C₁₀H₉FN₄O₂S.

6.1.2. General method for the synthesis of thiosemicarbazones (A4-6,

B4-6)

To a hot dispersion of A3/B3 (0.001M) in ethanol was added an

equimolar aqueous solution of sodium acetate. To this solution

further an equimolar ethanolic solution of various 5-substituted

isatin was added and the mixture was stirred while being heated on

a hot plate for 4e15 min, with immediate precipitation of a bulky

solid. The resultant precipitate was ﬁltered off and dried. The

product was recrystallized from 95% ethanol.

6.1.2.6. (3Z)-5-Methyl-1H-indole-2,3-dione 3-(N-methoxy thiosemi-

carbazone) B6. %Yield: 34.72; M.P: 186e188 ^ꢂC; IR (KBr, cm^ꢀ1) 1740

(amide stretch), 1625 (C]N stretch), 3010 (Aromatic CeH stretch),

3300 (amide NH stretch); ¹H NMR (DMSO-d₆, 400 MHz)

d 9.17 (s,

1H, thioamido), 8.87 (s, 1H, hydrazino), 7.89 (s, 1H at C₄of indolyl),

7.63 (s,1H at N₁of indolyl), 7.16 (d,1H at C₇of indolyl), 6.92 (d, 1H at

C₆of indolyl), 3.53 (s, 3H, CH₃of methoxy), 2.57 (s, 3H, methoxy);

6.1.2.1. (3Z)-5-Chloro-1H-indole-2,3-dione

semicarbazone) (A4). %Yield: 62.96; M.P: 210 ^ꢂC (d); ¹H NMR

(DMSO-d₆, 400 MHz) 7.34 (d,1H at C₆of indolyl), 7.42 (d,1H at C₇of

3-(N-hydroxy

thio-

d

¹³C NMR (DMSO-d₆, 400 MHz)

d 20.58 (CH₃), 65.37 (C of methoxy

indolyl), 7.61 (s,1H, NH of indolyl), 7.96 (s,1H at C₄of indolyl), 8.52 (s,

thiosemicarbazone), 113.37 (C₇of indolyl), 126.13 (C_4aof indolyl),

127.6 (C₆of indolyl), 129.33 (C₅of indolyl), 129.78 (C4 of indolyl),

133.13 (C₃of indolyl), 140 (C_7aof indolyl), 154.06 (C₂of indolyl),

158.93 (C of carbothioamido); Anal[calculated/found]. C, 49.99/

49.79; H, 4.58/4.54; N, 21.20/21.28; C₁₁H₁₂N₄O₂S.

1H, hydrazino), 9.47 (d, 1H, thioamido), 10.51 (d, 1H, hydroxyl); ¹³C

NMR (DMSO-d₆, 400 MHz)

d 115.73 (C_4aof indolyl), 117.17 (C₇of

indolyl), 126.97 (C₄of indolyl), 127.36 (C₅of indolyl), 132.14 (C₆of

indolyl), 133.18 (C₇of indolyl), 139.86 (C_7aof indolyl), 152.48 (C of

carbothioamido), 154.06 (C₂of indolyl); Anal[calculated/found]. C,

39.93/39.97; H, 2.61/2.57; N, 20.70/20.66C₉H₇ClN₄O₂S.

6.1.3. General method for the synthesis of N-mannich bases of

5-substituted-1H-indole-2,3-dione 3-(N-hydroxy/methoxy-

thiosemicarbazones) (A7e36, B7e36)

To an ethanolic solution of N-hydroxy/methoxy thio-

semicarbazones (0.00045 M) was added an equimolar amount of

6.1.2.2. (3Z)-5-Fluoro-1H-indole-2,3-dione 3-(N-hydroxy thiosemi-

carbazone) (A5). %Yield: 56.57; M.P:223e225 ^ꢂC; IR (KBr, cm^ꢀ1

)

1740 (amide stretch), 1625 (C]N stretch), 3010 (Aromatic CeH

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

115

various secondary amines, dissolved in ethanol or dimethyl sulfoxide.

To this mixture, an equimolar amount of 30% formaldehyde solution

was then added and thismixturewas then irradiated with microwave

of 455 W in a scientiﬁc microwave reactor accompanied by contin-

uous stirring, which was attained by means of a magnetic plate,

placed underneath the ﬂoor of the magnetic cavity and a Teﬂon-

coated magnetic bead in the reaction vessel, for approximately three

to 15 min. At the end of the reaction period, the product precipitated

out, which was ﬁltered off and dried. The resultant precipitate was

recrystallized from ethanol.

[calculated/found]. C, 53.71/53.53; H, 6.31/6.34; N, 20.88/20.95;

₁₅H₂₁N₅O₂S.

C

6.1.3.5. 2-[5-chloro-1-(1,4-oxazinan-4-ylmethyl)-2-oxo-1,2-dihydro-

3H-indol-3-yliden]-N-hydroxyhydrazine-1-carbothioamide (A13). %

Yield: 56.43; M.P: 46e48 ^ꢂC; IR (cm^ꢀ1; KBr) 3020, 2847, 1725, 1615,

1220, 852; ¹H NMR (DMSO-d₆, 400 MHz)

d 2.64 (t, 4H, C₃, C₅of

morpholinyl), 3.55 (t, 4H, C₂, C₆of morpholinyl), 5.67 (s, 2H,

-NCH₂N-), 6.91 (d, 1H, C₇of indolyl), 7.41 (d, 1H, C₆of indolyl), 8.04

(s, 1H, C₄of indolyl), 8.49 (s, 1H, hydrazine), 9.46 (d, 1H, thioamido),

10.51 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d 56.69 (2C,

6.1.3.1. 2-{5-chloro-1-[(dimethylamino)methyl]-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N hydroxyhydrazine-1-carbothioamide (A7). %

Yield: 97.05; M.P: 102e104 ^ꢂC; IR (cm^ꢀ1; KBr) 3030, 2850, 1730,

C₃, C₅of morpholinyl), 58.8 (2C, C₂, C₆of morpholinyl), 62.14 (-CH₂),

118.23 (C₇of indolyl), 124.88 (C₄of indolyl), 126.87 (C₅of indolyl),

127.58 (C_4aof indolyl), 130.12 (C₆of indolyl), 136.12 (C₃of indolyl),

146.91 (C_7aof indolyl), 152.48 (C of carbothioamido), 155.39 (C₂of

indolyl); Anal[calculated/found]. C, 45.47/45.65; H, 4.36/4.35; N,

18.94/18.88C₁₄H₁₆ClN₅O₃S.

1620, 1210, 850; ¹H NMR (DMSO-d₆, 400 MHz)

d 2.07 (s, 6H, dime-

thylamine), 5.92 (s, 2H, -NCH₂N-), 6.61 (d,1H, indolyl C₇), 6.92 (d,1H,

indolyl C₆), 7.35 (s,1H, indolyl C₄) 8.51 (s,1H, hydrazino), 9.48 (d,1H,

thioamido), 10.52 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d

42.9 (2C, eN(CH₃)₂), 66.33 (-CH₂), 118 (C₇of indolyl), 124.88 (C₄of

6.1.3.6. 2-(5-Fluoro-2-oxo-1-{[4-phenyltetrahydropyrazin-1(2H)-yl]

methyl}-1,2-dihydro-3H-indol-3-yliden)-N-hydroxyhydrazine-1-car-

bothioamide (A17). %Yield: 86.09; M.P: 78e80 ^ꢂC; ¹H NMR (DMSO-

indolyl), 126.87 (C₅of indolyl), 127.36 (C_4aof indolyl), 130.12 (C₆

of indolyl), 136.12 (C₃of indolyl), 148.65 (C_7aof indolyl), 152.48 (C of

carbothioamido), 155.39 (C₂of indolyl); Anal[calculated/found]. C,

43.97/43.79; H, 4.30/4.26; N, 21.37/21.35; C₁₂H₁₄ClN₅O₂S.

d₆, 400 MHz) d 3.11 (t, 4H, C₂, C₆of piperazinyl), 3.18 (t, 4H, C₃, C₅of

piperazinyl), 6.50 (d, 1H, C₇of indolyl), 6.63 (d, 2H, C₂, C₆of phenyl),

6.81 (m, 1H, C₄of phenyl), 7.25 (d, 2H, C₃, C₅of phenyl), 7.30 (d, 1H,

C₆of indolyl), 7.63 (s, 1H, C₄of indolyl), 8.51 (s, 1H, hydrazine), 9.47

(d, 1H, thioamido), 10.52 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆,

6.1.3.2. 2-{1-[(Dimethylamino)methyl]-5-methyl-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N-hydroxyhydrazine-1-carbothioamide (A9). %

Yield: 95.85; M.P: 110 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d

2.13 (s,

400 MHz) d 48.17 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of

6H, dimethylamino), 2.65 (s, 3H, CH₃at C₅of indolyl), 5.92 (s, 2H,

-NCH₂N-), 7.11 (d, 1H, C₇of indolyl), 7.23 (d, 1H, C₆of indolyl), 7.47

(s, 1H, C₄of indolyl), 8.52 (s, 1H, hydrazine), 9.48 (d, 1H, thioamido),

piperazinyl), 62.25 (-CH₂), 113.91 (C₄of indolyl), 115.07 (C₆of

indolyl),116.35 (2C, C₂, C₆of phenyl), 117.69 (C₇of indolyl),120.3 (C₄

of phenyl),128.19 (C_4aof indolyl), 129.1 (2C, C₃, C₅of phenyl), 133.61

(C₃of indolyl), 145.62 (C_7aof indolyl), 147.57 (C₅of indolyl), 150.73

(C₁of phenyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl);

Anal[calculated/found]. C, 56.06/55.84; H, 4.94/4.96; N, 19.61/

19.57C₂₀H₂₁FN₆O₂S.

10.51 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d 20.58 (CH₃

at C₅of indolyl), 42.9 (2C, -N(CH₃)₂), 66.33 (eCH₂), 115.45 (C₇of

indolyl), 124.39 (C₅of indolyl), 126.14 (C₆of indolyl), 128.92 (C_4a

of indolyl), 129.42 (C₄of indolyl), 136.07 (C₃of indolyl), 149.99 (C_7a

of indolyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl);

Anal[calculated/found]. C, 50.80/50.66; H, 5.57/5.53; N, 22.78/

22.85; C₁₃H₁₇N₅O₂S.

6.1.3.7. N-Hydroxy-2-(5-methyl-2-oxo-1-{[4-phenyl-

tetrahydropyrazin-1(2H)-yl]methyl}-1,2-dihydro-3H-indol-3-yliden)

hydrazine-1-carbothioamide (A18). %Yield: 79.93; M.P: 68e70 ^ꢂC;

IR (cm^ꢀ1; KBr) 3030, 2850, 1731, 1622, 1210, 845; ¹H NMR (DMSO-

6.1.3.3. 2-{1-[(Diethylamino)methyl]-5-ﬂuoro-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N-hydroxyhydrazine-1-carbothioamide (A11).

%Yield: 79.90; M.P: 80e82 ^ꢂC; IR (cm^ꢀ1; KBr) 3040, 2855, 1735,

d₆, 400 MHz)

d 2.26 (s, 3H, CH₃), 3.15 (t, 4H, C₂, C₆of piperazinyl),

3.17 (t, 4H, C₃, C₅of piperazinyl), 5.13 (s, 2H, -NCH₂N-), 6.41 (d, 2H,

C₂, C₆of phenyl), 6.71 (d, 1H, C₇of indolyl), 6.77 (m, 1H, C₄of

phenyl), 7.11 (d, 1H, C₆of indolyl), 7.35 (d, 2H, C₃, C₅of phenyl), 7.76

(s,1H, C₄of indolyl), 8.52 (s, 1H, hydrazine), 9.47 (d, 1H, thioamido),

1621, 1215, 843; ¹H NMR (DMSO-d₆, 400 MHz)

d 1.03 (t, 6H,

(CH₂CH₃)₂), 2.59 (q, 4H, (CH₂CH₃)₂), 5.94 (s, 2H, eNCH₂Ne), 6.65 (d,

1H, C₇of indolyl), 7.22 (d, 1H, C₆of indolyl), 7.83 (s, 1H, C₄of

indolyl), 8.52 (s, 1H, hydrazine), 9.49 (d, 1H, thioamido), 10.45 (d,

10.53 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d 20.58 (CH₃

1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d

12.01 (2C of CH₃

at C₅of indolyl), 48.17 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of

piperazinyl), 62.25 (-CH₂), 115.89 (C₇of indolyl), 116.35 (2C, C₂, C₆

of phenyl), 120.3 (C₄of phenyl), 124.39 (C₅of indolyl), 126.14 (C₆of

indolyl), 129.1 (2C, C₃, C₅of phenyl), 129.37 (C_4aof indolyl), 129.42

(C4 of indolyl), 136.07 (C3 of indolyl), 148.71 (C7a of indolyl), 150.73

(C₁of phenyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl);

Anal[calculated/found]. C, 59.51/59.41; H, 5.70/5.70; N,19.80/19.80;

C₂₁H₂₄N₆O₂S.

of eN(C₂H₅)₂), 42.89 (2C of CH₂of eN(C₂H₅)₂), 61.91 (-CH₂), 111.71

(C₆of indolyl), 113.91 (C₄of indolyl), 117.76 (C₇of indolyl), 127.75

(C_4aof indolyl), 133.61 (C₃of indolyl), 146.12 (C_7aof indolyl), 147.57

(C₅of indolyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl);

Anal[calculated/found]. C, 49.54/49.37; H, 5.35/5.33; N, 20.64/

20.69; C₁₄H₁₈FN₅O₂S.

6.1.3.4. 2-{1-[(Diethylamino)methyl]-5-methyl-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N-hydroxyhydrazine-1-carbothioamide (A12). %

6.1.3.8. 2-(1-{[4-(4-Chlorophenyl)tetrahydropyrazin-1(2H)-yl]

methyl}-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-hydrox-

Yield: 71.47; M.P: 114e116 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d 1.08

(t, 6H, (CH₂CH₃)₂), 2.14 (s, 3H, CH₃) 2.53 (q, 4H, (CH₂CH₃)₂), 5.87 (s,

2H, -NCH₂N-), 6.47 (d, 1H, C₇of indolyl), 7.19 (d, 1H, C₆of indolyl),

7.64 (s, 1H, C₄of indolyl), 8.51 (s, 1H, hydrazine), 9.47 (d, 1H, thio-

amido), 10.43 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

yhydrazine-1-carbothioamide

178e182 ^ꢂC; IR (cm^ꢀ1; KBr) 3015, 2853, 1727, 1625, 1220, 853; ¹H

NMR (DMSO-d₆, 400 MHz) 2.56 (s, 3H, CH₃), 3.14 (t, 4H, C₂, C₆of

piperazinyl), 3.18 (t, 4H, C₃, C₅of piperazinyl), 5.73 (s, 2H, -NCH₂N-),

6.34 (d, 2H, C₂, C₆of phenyl), 6.63 (d, 1H, C₇of indolyl), 6.81 (d,

2H, C₃, C₅of phenyl), 7.54 (s,1H, C₄of indolyl), 8.53 (s, 1H, hydra-

zine), 9.47 (d, 1H, thioamido), 10.53 (d, 1H, hydroxyl); ¹³C NMR

(A21). %Yield:

76.34;

M.P:

d

12.03 (2C of CH₃of eN(C₂H₅)₂), 20.58 (CH₃at C₅of indolyl), 42.89

(2C of CH₂of eN(C₂H₅)₂), 61.91 (eCH₂), 115.07 (C₇of indolyl),

124.39 (C₅of indolyl), 124.57 (C₆of indolyl), 128.93 (C_4aof

indolyl), 129.42 (C₄of indolyl), 136.07 (C₃of indolyl), 149.59 (C_7aof

indolyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl); Anal

(DMSO-d₆, 400 MHz)

d 20.58 (CH₃at C₅of indolyl), 48.17 (2C, C₃, C₅

of piperazinyl), 51.73 (2C, C₂, C₆of piperazinyl), 62.25 (-CH₂-),

116

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

115.89 (C₇of indolyl), 124.39 (C₅of indolyl), 124.84 (2C, C₂, C₆of

Phenyl), 126.14 (C₆of indolyl), 127.93 (2C, C₃, C₅of Phenyl), 129.37

(C_4aof Indolyl), 129.42 (C₄of indolyl), 129.95 (C₄of Phenyl), 136.07

(C₃of indolyl), 146.74 (C₁of Phenyl), 148.71 (C_7aof Indolyl), 152.48

(C of carbothioamido), 155.39 (C₂of indolyl); Anal[calculated/

found]. C, 54.96/55.16; H, 5.05/5.03; N, 18.31/18.27C₂₁H₂₃ClN₆O₂S.

175.8 (C₄of quinolinyl); Anal[calculated/found]. C, 55.76/55.57; H,

4.85/4.86; N, 16.86/16.89C₂₇H₂₈FN₇O₅S.

6.1.3.12. 1-Cyclopropyl-6-ﬂuoro-7-[4-(5-ﬂuoro-3-{(Z)-2-[(hydrox-

yamino)carbothioyl]hydrazono}-2-oxo-2,3-dihydro-1H-indol-1-yl)

hexahydropyrazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic

acid (A29). %Yield: 90.62; M.P: 230(d)^ꢂC; IR (cm^ꢀ1; KBr) 3455,

3040, 2835, 1728, 1615, 1225, 846; ¹H NMR (DMSO-d₆, 400 MHz)

6.1.3.9. 2-(5-Chloro-1-{[4-[4-(methyloxy)phenyl]tetrahydropyrazin-

1(2H)-yl]methyl}-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-hydrox-

yhydrazine-1-carbothioamide (A22). %Yield: 58.32; M.P: 140 ^ꢂC; IR

(cm^ꢀ1; KBr) 3025, 2845, 1720, 1622, 1230, 848; ¹H NMR (DMSO-d₆,

d

0.39 (m, 4H, CH₂of cyclopropyl), 2.51 (t, 1H, CH of cyclopropyl),

3.11 (t, 4H, C₂, C₆of piperazinyl), 3.17 (t, 4H, C₃, C₅of piperazinyl),

5.09 (s, 2H, methylene), 6. 41 (s, 1H at C8 of quinolinyl), 6.61 (d, 1H

at C₇of indolyl), 6.97 (s, 1H at C₅of quinolinyl), 7.18 (d, 1H at C₆of

indolyl), 7.51 (s, 1H at C₂of quinolinyl), 7.73 (s, 1H at C₄of indolinyl),

8.53 (s, 1H, hydrazino), 9.46 (d, 1H, thioamido), 10.51 (d, 1H,

400 MHz)

d 3.15 (t, 4H, C₂, C₆of piperazinyl), 3.21 (t, 4H, C₃, C₅of

piperazinyl), 3.77 (s, 3H, 4-methoxyphenyl) 5.16 (s, 2H, -NCH₂N-),

6.16(d, 2H, C₂, C₆of phenyl), 7.43 (d,1H, C₇of indolyl), 6.62 (d, 2H, C₃,

C₅of phenyl), 6.89 (d,1H, C₆of indolyl), 8.14 (s,1H, C₄of indolyl), 8.53

(s,1H, hydrazino), 9.46 (d,1H, thioamido),10.51 (d,1H, hydroxyl); ¹³C

hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d 7.7 (2C, CH₂of cyclo-

propyl), 30.6 (CH of cyclopropyl), 50.02 (2C, C₃, C₅of piperazinyl),

51.73 (2C, C₂, C₆of piperazinyl), 62.25 (-CH₂-), 99.43 (C₈of quino-

linyl), 103.6 (C₅of quinolinyl), 107.6 (C₃of quinolinyl), 112.3 (C_5aof

quinolinyl), 113.91 (C₄of indolyl), 115.07 (C₆of indolyl), 117.69 (C₇of

indolyl), 128.19 (C_4aof indolyl), 133.03 (C_8aof indolyl), 133.61 (C₃of

indolyl), 139.76 (C₇of quinolinyl), 140.9 (C₂of quinolinyl), 145.62

(C_7aof indolyl), 147.57 (C₅of indolyl), 148.43 (C₆of quinolinyl),

152.48 (C of carbothioamido), 155.39 (C₂of indolyl), 161.9 (C of

COOH), 177.5 (C₄of quinolinyl); Anal[calculated/found]. C, 54.27/

54.08; H, 4.22/4.23; N, 16.41/16.35C₂₇H₂₅F₂N₇O₅S.

NMR (DMSO-d₆, 400 MHz) d 48.17 (C₃, C₅of piperazinyl), 51.73 (2C,

C₂, C₆of piperazinyl), 55.2 (C of methoxy phenyl), 62.25 (-CH₂-),

112.82 (2C, C₃, C₅of Phenyl), 118.45 (C₇of indolyl), 124.44 (2C, C₂, C₆

of Phenyl), 124.88 (C₄of indolyl), 126.87 (C₅of indolyl), 127.8 (C_4aof

indolyl), 130.12 (C₆of indolyl), 136.12 (C₃of indolyl), 142.19 (C₁of

Phenyl),146.83 (C_7aof indolyl),152.48 (C of carbothioamido),155.39

(C₂of indolyl), 157.24 (C₄of Phenyl); Anal[calculated/found]. C,

53.10/52.91; H, 4.88/4.89; N, 17.69/17.63C₂₁H₂₃ClN₆O₃S.

6.1.3.10. N-Hydroxy-2-(5-methyl-1-{[4-[4-(methyloxy)phenyl]tetra-

hydropyrazin-1(2H)-yl]methyl}-2-oxo-1,2-dihydro-3H-indol-3-yli-

den)hydrazine-1-carbothioamide (A24). %Yield: 88.66; M.P:

6.1.3.13. 7-[4-(5-chloro-3-{(Z)-2-[(hydroxyamino)carbothioyl]hydra-

zono}-2-oxo-2,3-dihydro-1H-indol-1-yl)-3-methylhexahydropyr-

azin-1-yl]-1-ethyl-6,8-diﬂuoro-4-oxo-1,4-dihydroquinoline-3-

216e218 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d 2.83 (s, 3H, CH₃), 3.23

carboxylic acid (A31). %Yield: 88.59; M.P: 226e228 ^ꢂC; IR (cm^ꢀ1

;

(t, 4H, C₂, C₆of piperazinyl), 3.34 (t, 4H, C₃, C₅of piperazinyl), 3.65

(s, 3H, 4-methoxyphenyl) 4.97 (s, 2H, -NCH₂N-), 6.23(d, 2H, C₂, C₆of

phenyl), 7.34 (d,1H, C₇of indolyl), 6.81 (d, 2H, C₃, C₅of phenyl), 6.89

(d, 1H, C₆of indolyl), 7.83 (s,1H, C₄of indolyl), 8.51 (s, 1H, hydra-

zine), 9.45 (d, 1H, thioamido), 10.53 (d, 1H, hydroxyl); ¹³C NMR

KBr) 3500, 3020, 2841, 1725, 1621, 1217, 845; ¹H NMR (DMSO-d₆,

400 MHz) 0.94 (d, 3H, CH₃at C3 of piperazinyl), 1.18 (t, 3H of CH₃

d

of C₂H₅), 2.85 (d, 2H at C₂of piperazinyl), 3.14 (t, 2H at C₆of

piperazinyl), 3.21 (m, 1H at C₃of piperazinyl), 3.57 (t, 2H at C₅of

piperazinyl), 4.32 (q, 2H of C₂H₅), 5.13 (s, 2H of methylene), 6.91 (d,

1H at C₇of indolyl), 7.13 (s,1H at C₅of quinolinyl), 7.41 (d,1H at C₆of

indolyl), 7.52 (s, 1H at C₂of quinolinyl), 7.95 (s, 1H at C₄of indolyl),

8.52 (s, 1H, hydrazino), 9.47 (d, 1H, thioamido), 10.51 (d, 1H,

(DMSO-d₆, 400 MHz) d 20.58 (CH₃), 48.17 (2C, C₃, C₅of Piperazinyl),

51.73 (2C, C₂, C₆of Piperazinyl), 55.2 (CH₃of 4-methoxy phenyl),

62.25 (-CH₂-), 112.82 (2C, C₃, C₅of phenyl), 115.89 (C₇of indolyl),

124.39 (C5 of indolyl), 124.44 (2C, C₂, C₆of indolyl), 126.14 (C₆of

indolyl), 129.37 (C_4aof indolyl), 129.42 (C₄of indolyl), 136.07 (C₃

of indolyl), 142.19 (C₁of phenyl), 148.71 (C_7aof indolyl), 152.48 (C of

carbothioamido), 155.39 (C₂of indolyl), 157.24 (C₄of Phenyl); Anal

[calculated/found]. C, 58.13/57.92; H, 5.77/5.79; N, 18.49/18.54;

C₂₂H₂₆N₆O₃S.

hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

d 15.7 (CH₃of C2H5 at N1

of quinolinyl), 18.12 (CH3 at C3 of piperazinyl), 46.41 (CH2 of C2H5

at N1 of quinolinyl), 51.26 (C₄of quinolinyl), 52.38 (C₆of piper-

azinyl), 52.88 (C₅of piperazinyl), 57.64 (C₃of piperazinyl), 60.04

(-CH₂-), 62.35 (C₂of piperazinyl), 103.89 (C₃of quinolinyl), 108.28

(C₅of quinolinyl), 112.89 (C₇of quinolinyl), 118.45 (C₇of indolyl),

119.87 (C_4aof quinolinyl), 124.88 (C₄of indolyl), 126.87 (C₅of

indolyl), 127.8 (C_4aof indolyl), 130.12 (C₆of indolyl), 130.85 (C_8aof

quinolinyl), 136.12 (C₃of indolyl), 139.48 (C₈of quinolinyl), 142.58

(C₆of quinolinyl), 145.61 (C₂of quinolinyl), 146.49 (C_7aof indolyl),

152.48 (C of carbothioamido), 155.39 (C₂of indolyl), 169.04 (C of

COOH); Anal[calculated/found]. C, 51.15/51.29; H, 4.13/4.15; N,

15.46/15.48; C₂₇H₂₆ClF₂N₇O₅S.

6.1.3.11. 1-Ethyl-6-ﬂuoro-7-[4-(3-{(Z)-2-[(hydroxyamino)carbo-

thioyl]hydrazono}-5-methyl-2-oxo-2,3-dihydro-1H-indol-1-yl)hex-

ahydropyrazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

(A27). %Yield: 47.33; M.P: 180e182 ^ꢂC (d); ¹H NMR (DMSO-d₆,

400 MHz)

d 1.21 (t, 3H, CH₃of C₂H₅), 2.37 (s, 3H, methyl at C₅of

indole), 3.11 (t, 4H, C₂, C₆of piperazinyl), 3.15 (t, 4H, C₃, C₅of piper-

azinyl), 4.26 (q, 2H, CH₂of C₂H₅), 5.16 (s, 2H, methylene CH₂), 6.21 (s,

1H at C₈of quinolinyl), 6.73 (d,1H at C₇of indolyl), 7.14 (d,1H at C₆of

indolyl), 7.25 (s, 1H at C₅of quinolinyl), 7.29 (s, 1H at C₄of indolyl),

7.35 (s, 1H at C₂of quinolinyl), 8.52 (s, 1H, hydrazino), 9.47 (d, 1H,

thioamido), 10.51 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

6.1.3.14. 1-Ethyl-6,8-diﬂuoro-7-[4-(3-{(Z)-2-[(hydroxyamino)carbo-

thioyl]hydrazono}-5-methyl-2-oxo-2,3-dihydro-1H-indol-1-yl)-

3-methylhexahydropyrazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-

carboxylic acid (A33). %Yield: 81.22; M.P: 140e144 ^ꢂC; ¹H NMR

d

14.4 (CH₃of C₂H₅at N₁of quinolinyl), 20.58 (CH₃at C₅of indolyl),

(DMSO-d₆, 400 MHz) d 0.96 (d, 3H, CH₃at C₃of piperazinyl), 1.17 (t,

49.01 (CH₂of C₂H₅at N₁of quinolinyl), 50.02 (2C, C₃, C₅of piper-

azinyl), 51.73 (2C, C₂, C₆of piperazinyl), 62.25 (-CH₂-), 106.84 (C₈of

quinolinyl),107.2 (C₃ofquinolinyl),111.2(C₅of quinolinyl),115.89 (C₇

of indolyl), 119.8 (C_4aof quinolinyl), 124.39 (C₅of indolyl),126.14 (C₆

of indolyl),129.37 (C_4aof indolyl),129.42 (C₄of indolyl),136.07 (C₃of

indolyl),136.9 (C_8aof quinolinyl),143.37 (C7 of quinolinyl),148.4 (C2

of quinolinyl), 148.71 (C7a of indolyl), 152.48 (C of carbothioamido),

152.83 (C₆of quinolinyl), 155.39 (C₂of indolyl), 165.6 (C of COOH),

3H of CH₃of C₂H₅), 2.41 (s, 3H, methyl at C₅of indolyl), 2.87 (d, 2H

at C₂of piperazinyl), 3.13 (t, 2H at C₆of piperazinyl), 3.19 (m, 1H at

C₃of piperazinyl), 3.58 (t, 2H at C₅of piperazinyl), 4.34 (q, 2H of

C₂H₅), 5.15 (s, 2H of methylene), 6.92 (d, 1H at C₇of indolyl), 7.15 (s,

1H at C₅of quinolinyl), 7.43 (d, 1H at C₆of indolyl), 7.51 (s, 1H at C₂

of quinolinyl), 7.93 (s, 1H at C₄of indolyl), 8.51 (s, 1H, hydrazino),

9.47 (d, 1H, thioamido), 10.52 (d, 1H, hydroxyl); ¹³C NMR (DMSO-d₆,

400 MHz)

d 15.7 (CH₃of C₂H₅), 18.12 (CH₃at C₃of piperazinyl),

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

117

20.58 (CH₃at C₅of indolyl), 46.41 (CH₂of C₂H₅), 51.26 (C₄of qui-

6.1.3.18. 2-[5-methyl-1-(1,4-oxazinan-4-ylmethyl)-2-oxo-1,2-dihy-

nolinyl), 52.38 (C₆of piperazinyl), 52.88 (C₅of piperazinyl), 57.64

(C₃of piperazinyl), 60.04 (-CH₂-), 62.35 (C₂of piperazinyl), 112.89

(C₇of quinolinyl), 115.89 (C₇of indolyl), 119.87 (C_4aof quinolinyl),

124.39 (C₅of indolyl), 126.14 (C₆of indolyl), 129.37 (C_4aof indolyl),

129.42 (C₄of indolyl), 136.07 (C₃of indolyl), 139.48 (C₈of quino-

linyl), 142.58 (C₆of quinolinyl), 145.61 (C₂of quinolinyl), 147.91 (C_7a

of indolyl), 152.48 (C of carbothioamido), 155.39 (C₂of indolyl),

169.04 (C of COOH); Anal[calculated/found]. C, 54.80/54.63; H,

4.76/4.75; N, 15.98/15.93C₂₈H₂₉F₂N₇O₅S.

dro-3H-indol-3-yliden]-N-(methyloxy)hydrazine-1-carbothioamide

(B15). %Yield: 75.70; M.P: 86e90 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d

2.43 (s, 3H, CH₃), 2.65 (t, 4H, C₃, C₅of morpholinyl), 3.56 (t, 4H, C₂,

C₆of morpholinyl), 3.66 (s, 3H, methoxyl), 5.17 (s, 2H, -NCH₂N-),

6.73 (d, 1H, C₇of indolyl), 7.13 (d, 1H, C₆of indolyl), 7.32 (s, 1H, C₄of

indolyl), 8.51 (s, 1H, hydrazino), 9.47 (s, 1H, NH proton); ¹³C NMR

(DMSO-d₆, 400 MHz) d 20.58 (CH₃at C₅of indolyl), 56.69 (2C, C₃, C₅

of morpholinyl), 58.8 (2C, C₂, C₆of morpholinyl), 62.14 (-CH₂-),

65.37 (CH₃of methoxythiosemicarb), 115.67 (C₇of indolyl), 124.39

(C₅of indolyl), 126.14 (C₆of indolyl), 129.23 (C_4aof indolyl), 129.42

(C₄of indolyl), 136.07 (C₃of indolyl), 148.79 (C_7aof indolyl), 155.39

(C₂of indolyl), 158.93 (C of carbothioamido); Anal[calculated/

found]. C, 52.88/53.09; H, 5.82/5.83; N, 19.27/19.21; C₁₆H₂₁N₅O₃S

6.1.3.15. 1-Cyclopropyl-6-ﬂuoro-7-[4-(3-{(Z)-2-[(hydroxyamino)car-

bothioyl]hydrazono}-5-methyl-2-oxo-2,3-dihydro-1H-indol-1-yl)-3-

methylhexahydropyrazin-1-yl]-8-(methyloxy)-4-oxo-1,4-dihy-

droquinoline-3-carboxylic acid (A36). %Yield: 77.75; M.P: 70 ^ꢂC; IR

(cm^ꢀ1; KBr) 3458, 3035, 2851, 1732, 1618,1210, 852; NMR (DMSO-d₆,

6.1.3.19. 2-(5-Chloro-2-oxo-1-{[4-phenyltetrahydropyrazin-1(2H)-

yl]methyl}-1,2-dihydro-3H-indol-3-yliden)-N-(methyloxy)hydrazine-

1-carbothioamide (B16). %Yield: 93.33; M.P: 80e82 ^ꢂC; ¹H NMR

400 MHz) d 0.39 (m, 4H, CH₂of cyclopropyl), 0.94 (s, 3H, CH₃at C₃of

piperazinyl), 2.41 (s, 3H, methyl at C₅of indolyl), 2.44 (t, 1H, CH of

cyclopropyl), 2.89 (d, 2H at C₂of piperazinyl), 3.14 (t, 2H at C₆

of piperazinyl), 3.18 (m, 1H at C₃of piperazinyl), 3.56 (t, 2H at C₅of

piperazinyl), 3.61 (s, 3H, methoxy at C₈of quinolinyl), 5.15 (s, 2H of

methylene), 6.71 (d, 1H at C₇of indolyl), 7.08 (d, 1H at C₆of indolyl),

7.30 (s, 1H at C₄of indolyl), 7.34 (s,1H, C₅of quinolinyl), 7.41 (s,1H, C₂

ofquinolinyl), 8.51 (s,1H, hydrazino), 9.46 (d,1H, thioamido),10.53(d,

(DMSO-d₆, 400 MHz) d 3.15 (t, 4H, C₂, C₆of piperazinyl), 3.18 (t, 4H, C₃,

C₅of piperazinyl), 3.43 (s, 3H, methoxyl), 5.15 (s, 2H, -NCH₂N-), 6.73

(d, 2H, C₂, C₆of phenyl), 6.83 (d, 1H, C₄of phenyl), 6.81(d, 1H, C₇of

indolyl), 7.21 (m, 2H, C₃, C₅ofphenyl), 7.25 (d,1H, C₆of indolyl), 8.13(s,

1H, C₄of indolyl), 8.53 (s, 1H, hydrazino), 9.46 (s, 1H, hydroxyl); ¹³

C

NMR (DMSO-d₆, 400 MHz) d 48.17 (2C, C₃, C₅of piperazinyl), 51.73 (2C,

1H, hydroxyl); ¹³C NMR (DMSO-d₆, 400 MHz)

cyclopropyl), 18.12 (CH₃at C₃of piperazinyl), 20.58 (CH₃at

d

7.7 (2C, CH₂of

of

C₂, C₆of piperazinyl), 62.25 (-CH₂-), 65.37 (CH₃of methoxy thiosem),

116.35 (2C, C₂, C₆of phenyl), 118.45 (C₇of indolyl), 120.3 (C₄of Ph),

124.88 (C₄of indolyl), 126.87 (C₅of indolyl), 127.88 (C_4aof

indolyl), 129.1 (2C, C₃, C₅of Phenyl), 130.12 (C₆of indolyl), 136.12 (C₃

of indolyl), 146.83 (C_7aof Indolyl), 150.73 (C₁of Phenyl), 155.39 (C₂of

indolyl), 158.93 (C of carbothioamido); Anal[calculated/found]. C,

54.96/55.14; H, 5.05/5.03; N, 18.31/18.36; C₂₁H₂₃ClN₆O₂S.

C5

indolyl), 31.97 (CH of cyclopropyl), 50.88 (C₆of piperazinyl), 52.88 (C₅

of piperazinyl), 57.64 (C₃of piperazinyl), 57.77 (methoxy C at C₈of

quinolinyl), 60.04 (-CH₂-), 60.85 (C₂of piperazinyl), 115.89 (C₇of

indolyl), 122.43 (C_4aof quinolinyl), 122.88 (C_8aof quinolinyl), 124.39

(C₅of indolyl),126.14 (C₆of indolyl),129.37 (C_4aof indolyl),129.42 (C₄

of indolyl), 132.29 (C₇of quinolinyl), 136.07 (C₃of indolyl), 142.76 (C8

of quinolinyl),146.39 (C2 of quinolinyl),147.91 (C7a of indolyl),152.48

(C of carbothioamido),155.07 (C₆of quinolinyl),155.39 (C₂of indolyl),

163.68 (C of COOH),174.28 (C₄of quinolinyl); Anal[calculated/found].

C, 56.50/56.68; H, 5.06/5.08; N, 15.38/15.34C₃₀H₃₂FN₇O₆S.

6.1.3.20. 2-(5-Chloro-1-{[4-(4-chlorophenyl)

(2H)-yl]methyl}-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-(methyl-

oxy)hydrazine-1-carbothioamide (B19). %Yield: 74.26; M.P:

208e210 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

3.17 (t, 4H, C₂, C₆of

tetrahydropyrazin-1

d

piperazinyl), 3.20 (t, 4H, C₃, C₅of piperazinyl), 3.51 (s, 3H,

methoxyl), 5.13 (s, 2H, -NCH₂N-), 6.65 (d, 2H, C₂, C₆of phenyl), 6.79

(d, 1H, C₇of indolyl), 6.82 (m, 2H, C₃, C₅of phenyl), 7.34 (d, 1H, C₆of

indolyl), 8.07 (s, 1H, C₄of indolyl), 8.53 (s, 1H, hydrazino), 9.48 (s,

6.1.3.16. 2-{1-[(Dimethylamino)methyl]-5-ﬂuoro-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N-(methyloxy)hydrazine-1-carbothioamide

(B8). %Yield: 82.45; M.P: 56e58 ^ꢂC; IR (cm^ꢀ1; KBr) 3020, 2847,

1726, 1615, 1205, 854; ¹H NMR (DMSO-d₆, 400 MHz)

d

2.45 (s, 6H,

1H, NH proton); ¹³C NMR (DMSO-d₆, 400 MHz)

d 48.17 (2C, C₃, C₅of

dimethylamino), 3.67 (s, 3H, methoxyl), 5.14 (s, 2H, -NCH₂N-), 6.63

(d, 1H, C₇of indolyl), 7.31 (d, 1H, C₆of indolyl), 7.82 (s, 1H, C₄of

indolyl), 8.51 (s, 1H, hydrazino), 9.48 (s, 1H. NH proton); ¹³C NMR

pip), 51.73 (2C, C₂, C₆of piperazinyl), 62.25 (-CH₂-), 65.37 (CH₃

of methoxy thiosem), 118.45 (C₇of indolyl), 124.84 (2C, C₂, C₆of

phenyl), 124.88 (C₄of indolyl), 126.87 (C₅of indolyl), 127.88 (C_4aof

Indolyl), 127.93 (2C, C₃, C₅of indolyl), 129.95 (C₄of phenyl), 130.12

(C₆of indolyl), 136.12 (C₃of indolyl), 146.74 (C₁of phenyl), 146.83

(C_7aof indolyl), 155.39 (C₂of indolyl), 158.93 (C of carbothioamido);

Anal[calculated/found]. C, 51.12/50.96; H, 4.49/4.49; N, 17.03/17.06;

C₂₁H₂₂Cl₂N₆O₂S.

(DMSO-d₆, 400 MHz)

d 42.9 (2C, -N(CH₃)₂), 65.37 (CH₃of methoxy

thiosemicarbazone), 66.33 (-CH₂-), 113.91 (C₄of indolyl), 115.07 (C₆

of indolyl),118.13 (C₇of indolyl),127.82 (C_4aof indolyl),133.61 (C₃of

indolyl), 146.51 (C_7aof indolyl), 147.57 (C₅of indolyl), 155.39 (C2 of

indolyl), 158.93 (C of carbothioamido); Anal[calculated/found]. C,

47.99/47.85; H, 4.96/4.98; N, 21.52/21.55C₁₃H₁₆FN₅O₂S.

6.1.3.21. 2-(1-{[4-(4-Chlorophenyl)tetrahydropyrazin-1(2H)-yl]

methyl}-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-(methyl-

oxy)hydrazine-1-carbothioamide (B21). %Yield: 85.46; M.P: 140 ^ꢂC;

6.1.3.17. 2-{1-[(Diethylamino)methyl]-5-ﬂuoro-2-oxo-1,2-dihydro-

3H-indol-3-yliden}-N-(methyloxy)hydrazine-1-carbothioamide

(B11). %Yield: 83.50; M.P: 98e100 ^ꢂC; ¹H NMR (DMSO-d₆,

¹H NMR (DMSO-d₆, 400 MHz)

d 2.43 (s, 3H, CH₃), 3.18 (t, 4H, C₂, C₆

400 MHz)

d

1.17 (t, 6H -(CH₂CH₃)₂), 2.53 (q, 4H, -(CH₂CH₃)₂), 3.59 (s,

of piperazinyl), 3.21 (t, 4H, C₃, C₅of piperazinyl), 3.57 (s, 3H,

methoxyl), 5.09 (s, 2H, -NCH₂N-), 6.32 (d, 2H, C₂, C₆of phenyl), 6.69

(d, 1H, C₇of indolyl), 6.81 (m, 2H, C₃, C₅of phenyl), 7.06 (d, 1H, C₆of

indolyl), 7.37 (s, 1H, C₄of indolyl), 8.51 (s, 1H, hydrazino), 9.46 (s,

3H, methoxyl), 5.17 (s, 2H, -NCH₂N-), 6.57 (d, 1H, C₇of indolyl), 7.33

(d, 1H, C₆of indolyl), 7.76 (s, 1H, C₄of indolyl), 8.52 (s, 1H, hydra-

zino), 9.46 (s, 1H, NH proton); ¹³C NMR (DMSO-d₆, 400 MHz)

d

12.03 (2C, CH₃of eN(C₂H₅)₂), 42.89 (2C, CH₂of eN(C₂H₅)₂), 61.91

1H, NH proton); ¹³C NMR (DMSO-d₆, 400 MHz)

d 20.58 (CH₃at C₅of

(-CH₂-), 65.37 (CH₃of methoxy thiosemicarbazone), 111.71 (C₆of

indolyl), 113.91 (C₄of indolyl), 117.76 (C₇of indolyl), 127.83 (C_4a

of indolyl),133.61 (C₃of indolyl),146.12 (C_7aof indolyl),147.57 (C₅of

indolyl), 155.39 (C₂of indolyl), 158.93 (C of carbothioamido); Anal

[calculated/found]. C, 50.98/50.89; H, 5.70/5.68; N, 19.82/19.85;

C₁₅H₂₀FN₅O₂S.

indolyl), 48.17 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of

piperazinyl), 62.25 (-CH₂-), 65.37 (CH3 of methoxy thiosem), 115.89

(C₇of indolyl), 124.39 (C₅of indolyl), 124.84 (2C, C₂, C₆of phenyl),

126.14 (C₆of indolyl), 127.93 (2C, C₃, C₅of phenyl), 129.42(C₄of

indolyl), 129.44 (C_4aof indolyl), 129.95 (C₄of phenyl), 136.07 (C₃

of indolyl), 146.74 (C₁of phenyl), 148.71 (C_7aof indolyl), 155.39 (C₂

118

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

of indolyl), 158.93 (C of carbothioamido); Anal[calculated/found]. C,

55.86/56.05; H, 5.33/5.35; N, 17.77/17.73; C₂₂H₂₅ClN₆O₂S.

6.1.3.25. 1-Cyclopropyl-6-ﬂuoro-7-[4-{[5-methyl-3-((Z)-2 {[(methyl-

oxy)amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H) yl]methyl}

tetrahydropyrazin-1(2H)-yl]-4-oxo-1,4-dihydroquinoline-3-carbox-

ylic acid (B30). %Yield: 85.67; M.P: 150e154 ^ꢂC (d); ¹H NMR

6.1.3.22. 2-(5-Fluoro-1-{[4-[4-(methyloxy)phenyl]tetrahydropyr-

azin-1(2H)-yl]methyl}-2-oxo-1,2-dihydro-3H-indol-3-yliden)-N-

(methyloxy)hydrazine-1-carbothioamide (B23). %Yield: 69.35; M.P:

(DMSO-d₆, 400 MHz) d 0.43 (m, 4H, CH₂of cyclopropyl), 2.48 (s, 3H,

methyl at C₅of indolyl), 2.53 (t, 1H, CH of cyclopropyl), 3.11 (t, 4H,

C₂, C₆of piperazinyl), 3.18 (t, 4H, C₃, C₅of piperazinyl), 3.61 (s, 3H,

methoxy), 5.13 (s, 2H, methylene), 6. 43 (s, 1H at C₈of quinolinyl),

6.67 (d, 1H at C₇of indolyl), 6.95 (s, 1H at C₅of quinolinyl), 7.16 (d,

1H at C₆of indolyl), 7.54 (s,1H at C₂of quinolinyl), 7.72 (s,1H at C₄of

indolyl), 8.53 (s, 1H, hydrazine), 9.47 (s, 1H, NH proton), 11.34 (s, 1H,

116e118 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d 3.14 (t, 4H, C₂, C₆of

piperazinyl), 3.19 (t, 4H, C₃, C₅of piperazinyl), 3.59 (s, 3H, methoxyl),

3.71 (s, 3H, 4-methoxyl), 5.11 (s, 2H, -NCH₂N-), 6.15 (d, 2H, C₂, C₆of

phenyl), 6.47 (d,1H, C₇of indolyl), 6.57 (m, 2H, C₃, C₅of phenyl), 7.23

(d,1H, C₆of indolyl), 7.73 (s,1H, C₄of indolyl), 8.51 (s,1H, hydrazino),

9.47 (s, 1H, NH proton); ¹³C NMR (DMSO-d₆, 400 MHz)

d

48.17 (2C,

carboxylate); ¹³C NMR (DMSO-d₆, 400 MHz)

d 7.7 (2C, CH₂of

C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of phenyl), 55.2 (CH₃of 4-

methoxy phenyl), 62.25 (-CH₂-), 65.37 (CH₃of methoxy thiosem),

112.82 (2C, C₃, C₅of phenyl), 113.91 (C₄of indolyl), 115.07 (C₆of

indolyl), 117.69 (C₇of indolyl), 124.44 (2C, C₂, C₆of phenyl), 128.27

(C_4aof indolyl), 133.61 (C₃of indolyl), 142.19 (C₁of phenyl), 145.62

(C_7aof phenyl),147.57 (C₅of indolyl),155.39 (C₂of indolyl),157.24 (C₄

of phenyl), 158.93 (C of carbothioamido); Anal[calculated/found]. C,

55.92/55.78; H, 5.33/5.31; N, 17.78/17.82; C₂₂H₂₅FN₆O₃S.

cyclopropyl), 20.58 (CH₃at C₅of indolyl), 30.6 (CH of cyclopropyl),

50.02 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of piperazinyl),

62.25 (-CH₂-), 65.37 (CH₃of methoxy thiosem), 99.43 (C₈of qui-

nolinyl), 103.6 (C₅of quinolinyl), 107.6 (C₃of quinolinyl), 112.3 (C_4a

of quinolinyl), 115.89 (C₇of indolyl), 124.39 (C₅of indolyl), 126.14

(C₆of indolyl), 129.42 (C₄of indolyl), 129.44 (C_4aof indolyl), 136.07

(C₃of indolyl), 139.76 (C₇of quinolinyl), 140.9 (C₂of quinolinyl),

148.43 (C₆of quinolinyl), 148.71 (C_7aof quinolinyl), 155.39 (C₂of

indolyl), 158.93 (C of carbothioamido), 161.9 (C of COOH), 177.5 (C₄

of quinolinyl); Anal[calculated/found]. C, 57.32/57.23; H, 4.98/4.99;

N, 16.14/16.11; C₂₉H₃₀FN₇O₅S.

6.1.3.23. 1-Ethyl-6-ﬂuoro-7-[4-{[5-ﬂuoro-3-((Z)-2-{[(methyloxy)

amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H)-yl]methyl}tet-

rahydropyrazin-1(2H)-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic

acid (B26). %Yield: 93.50; M.P: 66e68 ^ꢂC; ¹H NMR (DMSO-d₆,

6.1.3.26. 1-Ethyl-6,8-diﬂuoro-7-[4-{[5-ﬂuoro-3-((Z)-2-{[(methyloxy)

amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H)-yl]methyl}-3-

methyltetrahydropyrazin-1(2H)-yl]-4-oxo-1,4-dihydroquinoline-3-

carboxylic acid (B32). %Yield: 72.90; M.P: 180 ^ꢂC (d); ¹H NMR

400 MHz)

d 1.21 (t, 3H, CH₃of C₂H₅), 3.13 (t. 4H, C₂, C₆of piper-

azinyl), 3.16 (t, 4H, C₃, C₅of piperazinyl), 3.62 (s, 2H, methylene),

4.31 (q, 2H, CH₂of C₂H₅), 6.23 (s, 1H, C₈of quinolinyl), 6.61 (s, 3H,

methoxy), 7.21 (s,1H, C₅of quinolinyl), 7.29 (d, 1H, C₆of indolyl),

7.31 (s, 1H, C₂of quinolinyl), 7.73 (s, 1H, C₄of indolyl), 8.52 (s, 1H,

hydrazine), 9.49 (s, 1H, NH proton), 10.53 (s, 1H, carboxylate); ¹³C

(DMSO-d₆, 400 MHz) d 0.94 (d, 3H, methyl at C₃of piperazinyl), 1.21

(t, 3H, CH₃of C₂H₅), 2.85 (d, 2H, C₂of piperazinyl), 3.14 (t, 2H, C₆of

piperazinyl), 3.21 (m, 1H, C₃of piperazinyl), 3.56 (t, 2H, C₅of

piperazinyl), 3.61 (s, 3H, methoxy), 4.32 (q, 2H, CH₂of C₂H₅), 5.13 (s,

2H, methylene), 6.57 (d, 1H, C₇of indolyl), 7.19 (s, 1H, C₅of quino-

linyl), 7.31 (d, 1H, C₆of indolyl), 7.52 (s, 1H, C₂of quinolinyl), 7.71 (s,

1H, C₄of indolyl), 8.51 (s, 1H, hydrazine), 9.46 (s, 1H, NH proton),

NMR (DMSO-d₆, 400 MHz)

d 14.4 (CH₃of C₂H₅at N₁of quinolinyl),

49.01 (CH₂of C₂H₅), 50.02 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂,

C₆of piperazinyl), 62.25 (-CH₂-), 65.37 (CH₃of methoxy thiosem),

106.84 (C₈of quinolinyl), 107.04 (C₃of quinolinyl), 111.2 (C₅of

quinolinyl),113.91 (C₄of indolyl),115.07 (C6 of indolyl),117.69 (C₇of

indolyl), 119.8 (C_4aof quinolinyl), 128.27 (C_4aof indolyl), 133.61 (C₃

of indolyl), 136.9 (C_8aof quinolinyl), 143.37 (C₇of quinolinyl), 145.62

(C_7aof indolyl),147.57 (C₅of indolyl),148.4 (C₂of quinolinyl),152.83

(C₆of quinolinyl), 155.39 (C₂of indolyl), 158.93 (C of carbothioa-

mido), 165.6 (C of COOH), 175.8 (C₄of quinolinyl); Anal[calculated/

found]. C, 54.08/54.23; H, 4.54/4.56; N,16.35/16.38; C₂₇H₂₇F₂N₇O₅S.

10.74 (s,1H, carboxylate); ¹³C NMR (DMSO-d₆, 400 MHz)

d 15.7 (CH₃

of C₂H₅), 18.12 (CH₃at C₃of piperazinyl), 46.7 (CH₂of C₂H₅), 52.38

(C₆of piperazinyl), 52.88 (C₅of piperazinyl), 57.64 (C₃of piper-

azinyl), 60.04 (-CH₂-), 62.35 (C₂of piperazinyl), 65.37 (C of methoxy

thiosem), 105.99 (C₅of quinolinyl), 107 (C₃of quinolinyl), 113.91 (C₄

of indolyl), 115.07 (C₆of indolyl), 117.69 (C₇of indolyl), 121.2 (C_4aof

quinolinyl), 126.39 (C₇of quinolinyl), 128.27 (C_4aof indolyl), 131.49

(C_8aof quinolinyl), 133.61 (C₃of indolyl), 140.88 (C₈of quinolinyl),

143.89 (C₆of quinolinyl), 145.28 (C_7aof indolyl), 147.57 (C₅of

indolyl), 151.01 (C₂of quinolinyl), 155.39 (C₂of indolyl), 158.93 (C of

carbothioamido), 165.03 (C of COOH), 174.2 (C₄of quinolinyl); Anal

[calculated/found]. C, 53.24/53.17; H, 4.47/4.48; N, 15.52/15.46;

C₂₈H₂₈F₃N₇O₅S.

6.1.3.24. 7-[4-{[5-chloro-3-((Z)-2-{[(methyloxy)amino]carbothioyl}

hydrazono)-2-oxo-1H-indol-1(2H)-yl]methyl}tetrahydropyrazin-1

(2H)-yl]-1-cyclopropyl-6-ﬂuoro-4-oxo-1,4-dihydroquinoline-3-

carboxylic acid (B28). %Yield: 72.53; M.P: 128e130 ^ꢂC (d); ¹H NMR

(DMSO-d₆, 400 MHz) d 0.39 (m, 4H, CH₂of cyclopropyl), 2.51 (t, 1H,

CH of cyclopropyl), 2.95 (t, 4H, C₃, C₅of piperazinyl), 3.13 (t, 4H, C₂,

C₆of piperazinyl), 3.61 (s, 3H, methoxy), 5.09 (s, 2H, methylene), 6.

43 (s, 1H at C₈of quinolinyl), 6.67 (d, 1H at C₇of indolyl), 6.95 (s, 1H

at C₅of quinolinyl), 7.16 (d, 1H at C₆of indolyl), 7.54 (s, 1H at C₂of

quinolinyl), 7.72 (s, 1H at C₄of indolyl), 8.51 (s, 1H, hydrazine) 9.46

(s, 1H, NH proton), 11.03 (s, 1H, carboxylate); ¹³C NMR (DMSO-d₆,

6.1.3.27. 1-Cyclopropyl-6-ﬂuoro-7-[4-{[5-ﬂuoro-3-((Z)-2-{[(methyl-

oxy)amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H)-yl]

methyl}-3-methyltetrahydropyrazin-1(2H)-yl]-8-(methyloxy)-4-oxo-

1,4-dihydroquinoline-3-carboxylic acid (B35). %Yield: 70.23; M.P:

88e90 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d 0.41 (m, 4H, CH₂of

400 MHz)

d

7.7 (2C, CH₂of cyclopropyl), 30.6 (CH of cyclopropyl),

cyclopropyl), 0.93 (d, 3H, methyl at C₃of piperazinyl), 2.45 (t,1H, CH

of cyclopropyl), 2.87 (d, 2H, C₂of piperazinyl), 3.12 (t, 2H, C₆of

piperazinyl), 3.21 (m, 1H, C₃of piperazinyl), 3.53 (t, 2H, C₅

of piperazinyl), 3.57 (s, 3H, methoxy), 3.61 (s, 3H, methoxy at C₈of

quinolinyl), 5.12 (s, 2H, methylene), 6.59 (d,1H, C₇of indolyl), 7.25 (d,

1H, C₆of indolyl), 7.31 (s, 1H, C₅of quinolinyl), 7.38 (s, 1H, C₂of

quinolinyl), 7.72 (s, 1H, C₄of indolyl), 8.52 (s, 1H, hydrazine), 9.47 (s,

1H, NH proton), 10.66 (s, 1H, carboxylate); ¹³C NMR (DMSO-d₆,

50.02 (2C, C₃, C₅of piperazinyl), 51.73 (2C, C₂, C₆of piperazinyl),

62.25 (-CH₂-), 65.37 (CH₃of methoxy thiosem), 99.43 (C₈of qui-

nolinyl), 103.6 (C₅of quinolinyl), 107.6 (C₃of quinolinyl), 112.3 (C_4a

of quinolinyl), 118.45 (C₇of indolyl), 124.88 (C₄of indolyl), 126.87

(C₅of indolyl), 127.88 (C_4aof indolyl), 130.12 (C₆of indolyl), 133.01

(C_8aof indolyl),136.12 (C₃of indolyl),139.76 (C₇of quinolinyl),140.9

(C₂of quinolinyl), 146.83 (C_7aof indolyl), 148.43 (C₆of quinolinyl),

155.39 (C₂of indolyl), 158.93 (C of carbothioamido) 161.9 (C of

COOH), 177.5 (C₄of quinolinyl); Anal[calculated/found]. C, 53.54/

53.71; H, 4.33/4.35; N, 15.61/15.64; C₂₈H₂₇ClFN₇O₅S.

400 MHz)

d 7.7 (2C, CH₂of cyclopropyl), 18.12 (CH₃at C₃of piper-

azinyl), 31.97 (CH of cyclopropyl), 50.88 (C₆of piperazinyl), 52.88 (C₅

of piperazinyl), 57.64 (C₃of piperazinyl), 57.77 (methoxy C at C₈of

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

119

quinolinyl), 60.04 (-CH₂-), 60.85 (C₂of piperazinyl), 65.37 (C of

methoxy thiosem), 102.56 (C₅of quinolinyl), 107.04 (C₃of quino-

linyl), 113.91 (C₄of indolyl), 115.07 (C₆of indolyl), 117.69 (C₇of

indolyl), 122.43 (C_4aof quinolinyl),122.88 (C_8aof quinolinyl), 128.27

(C_4aof indolyl), 132.29 (C₇of quinolinyl), 133.61 (C₃of indolyl),

142.76 (C₈of quinolinyl), 145.28 (C_7aof quinolinyl), 146.39 (C₂of

quinolinyl),147.57 (C₅of indolyl),155.07 (C₆of quinolinyl),155.39 (C₂

of indo),158.93 (C of carbothioamido),163.68 (C of COOH),174.28 (C₄

of quinolinyl); Anal[calculated/found]. C, 54.95/55.12; H, 4.77/4.78;

N, 14.95/14.98; C₃₀H₃₁F₂N₇O₆S.

albumin, an appropriate concentration of radiolabelled substrate [³H]

dGTP, 0.1 mM poly ( C)$oligo (dG) as the template/primer, 0.06%

Triton X-100, 10 L of inhibitor solution (containing various concen-

trations of compounds), and 1 L of RT preparation. The reaction

mixtures were incubated for 15 min at 37 ^ꢂC, after which 100

L of calf

thymus DNA (150 g/mL), 2 mL of Na₄P₂O₇(0.1 M in 1 M HCl), and

n

m

2 mL of trichloroacetic acid (10 %v/v) were added. The solutions were

kept on ice for 30 min, after which the acid-insoluble material was

washed and analyzed for radioactivity. For the experiments in which

50% inhibitory concentration (IC₅₀) of the test compounds was

determined, ﬁxed concentration of 2.5

m

[³H] dGTP was used [26].

6.1.3.28. 1-Cyclopropyl-6-ﬂuoro-7-[3-methyl-4-{[5-methyl-3-((Z)-2-

{[(methyloxy)amino]carbothioyl}hydrazono)-2-oxo-1H-indol-1(2H)-

yl]methyl}tetrahydropyrazin-1(2H)-yl]-8-(methyloxy)-4-oxo-1,4-

dihydroquinoline-3-carboxylic acid (B36). %Yield: 46.09; M.P:

6.2.3. In-vitro anti-tubercular activity in log phase cultures

All compounds were screened for their in vitro anti-mycobacterial

activity against log phase cultures of MTB in Middlebrook 7H11agar

medium supplementedwithOADC byagardilution method similarto

that recommended by the National Committee for Clinical Laboratory

Standards for the determination of MIC in triplicate. The MIC is

deﬁned as the minimum concentration of compound required to

produce complete inhibition of bacterial growth [27].

70e72 ^ꢂC; ¹H NMR (DMSO-d₆, 400 MHz)

d 0.41 (m, 4H, CH₂of

cyclopropyl), 0.93 (d, 3H, methyl at C₃of piperazinyl), 2.38 (s, 3H, C₅

of indolyl), 2.45 (t, 1H, CH of cyclopropyl), 2.87 (d, 2H, C₂of

piperazinyl), 3.12 (t, 2H, C₆of piperazinyl), 3.21 (m, 1H, C₃of

piperazinyl), 3.53 (t, 2H, C₅of piperazinyl), 3.57 (s, 3H, methoxy),

3.61 (s, 3H, methoxy at C₈of quinolinyl), 5.12 (s, 2H, methylene),

6.59 (d, 1H, C₇of indolyl), 7.25 (d, 1H, C₆of indolyl), 7.31 (s, 1H, C₅of

quinolinyl), 7.38 (s, 1H, C₂of quinolinyl), 7.72 (s, 1H, C₄of indolyl),

8.53 (s, 1H, hydrazine), 9.45 (s, 1H, NH proton), 11.37 (s, 1H,

6.2.4. In-vitro anti-tubercular activity in 6 week starved cultures

For starvation experiments, MTB cells were grown in Mid-

dlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol, 10%

(v/v) Middlebrook oleic acid-albumin-dextrose-catalase (OADC)

enrichment, and 0.025% (v/v) Tween 80 at 37 ^ꢂC with constant

rolling at 2 rpm until they reached an optical density at 600 nm of

w 0.6. The cells were then washed twice and re-suspended in

phosphate-buffered saline (PBS) at the same cell density. Cells

(50 mL of culture) were incubated at 37 ^ꢂC for an additional 6 weeks

in 1-L roller bottles. Compounds, dissolved in DMSO, were added to

either 1 mL PBS containing w 1 ꢃ 10⁷starved MTB cells at various

concentrations. Cultures were incubated in 15-mL conical tubes at

37 ^ꢂC with constant shaking for 7 days and then washed twice in

PBS before dilutions were plated on Middlebrook 7H11 plates

supplemented with 0.2% (v/v) glycerol, 10% (v/v) Middlebrook

OADC enrichment, and 0.025% (v/v) Tween 80, containing no

antibiotics. Bacterial growth was determined after incubation for 4

weeks at 37 ^ꢂC. The MIC is deﬁned as the minimum concentration

of compound which brings about complete inhibition of bacterial

growth. All values were determined in triplicate [28].

carboxylate); ¹³C NMR (DMSO-d₆, 400 MHz)

d 7.7 (2C, CH₂of

cyclopropyl), 18.12 (CH₃at C₃of piperazinyl), 20.58 (CH₃at C₅of

indolyl), 31.97 (CH of cyclopropyl), 50.88 (C₆of piperazinyl), 52.88

(C₅of piperazinyl), 57.64 (C₃of piperazinyl), 57.77 (methoxy C at C₈

of quinolinyl), 60.04 (-CH₂-), 60.85 (C₂of piperazinyl), 65.37 (C of

methoxy thiosem), 102.56 (C₅of quinolinyl), 107.03 (C₃of quino-

linyl), 115.89 (C₇of indolyl),122.43 (C_4aof quinolinyl), 122.88 (C_8aof

quinolinyl), 124.39 (C₅of indolyl), 126.14 (C₆of indolyl), 129.42 (C₄

of indolyl), 129.44 (C_4aof indolyl), 136.07 (C₃of indolyl), 132.29 (C₇

of quinolinyl), 142.76 (C₈of quinolinyl), 146.39 (C₂of quinolinyl),

147.91 (C_7aof indolyl), 155.07 (C₆of quinolinyl), 155.39 (C₂of

indolyl), 158.93 (C of carbothioamido), 163.68 (C of COOH), 174.28

(C₄of quinolinyl); Anal[calculated/found]. C, 57.13/56.97; H, 5.26/

5.28; N, 15.04/15.02; C₃₁H₃₄FN₇O₆S.

6.2. Biological activity

6.2.1. Anti-HIV activity

The compounds were evaluated for their anti-HIV activity and

cytotoxicity against replication of HIV-1 strain III_Bin MT-4 cells using

the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay method.. The MT-4 cells were grown in RPMI-1640 DM

(Dutch modiﬁcation) medium (Flow lab, Irvine Scotland), supple-

6.2.5. MTB ICL enzyme assay

Isocitrate lyase activity was determined at 37 ^ꢂC by measuring

the formation of glyoxylate-phenylhydrazone at 324 nm. The reac-

tion mixture contains 100

1.2 L of 1 mM magnesium chloride, 24

toethanol, 7 L of 4 mM phenylhydrazine hydrochloride, 6

50 mM trisodium isocitric acid and ICL enzyme (usually 3e6

This mixture is made up to 200 L with MilliQ water. At the end of

m

L of 0.5 mM potassium phosphate buffer,

L of 100 mM 2-mercep-

L of

L).

m

mented with 10% v/v heat-inactivated calf serum and 20

mg/mL

m

gentamicin (E. Merck Darmstadt, Germany). HIV-1 (IIIB) was obtained

from the culture supernatant of HIV-1 infected MT-4 cell lines and the

virus stocks were stored at ꢀ 70 ^ꢂC until used. Anti-HIV assays were

m

the 10th minute this reaction mixture is made up to 1 mL and UV

absorbance is measured at 324 nM which serves as a control. For the

carried out in microtitre plates ﬁlled with 100 mL of medium and 25 mL

volumes of compounds in triplicate so as to allow simultaneous

evaluation of theireffects on HIV-and mock-infected cells. 50 LofHIV

test compounds 3

candidate molecules 10

m

L of 100 mM 3-NPA was used and in case of the

m

mL of 10 mM concentration were added with

at 100 CCID₅₀(50% cell culture infective dose) medium were added to

either the HIV-infected or mock-infected part of the microtitre tray.

Cultureswereincubatedat37^ꢂCinahumidiﬁedatmosphereat5%CO₂

in atmosphere for ﬁve days. The viability of mock and HIV-infected

cells was examined spectrophotometrically to assess the anti-HIV

activity and cytopathic effect of the test compounds [26].

the above mentioned reaction mixture. At the end of the 10th

minute this reaction mixture is made up to 1 mL and UV absorbance

is measured at 324 nM which serves as a test. The % inhibition is

calculated by the formulae control absorbance minus test absor-

bance divided by control absorbance multiplied by 100.

6.3. Computational methodology

6.2.2. HIV-1 RT enzyme inhibition assay

The assay protocol involved a reaction mixture (50

of 50 mM tris-HCl (pH 7.8), 5 mM dithiothreitol, 30 mM glutathione,

50 M EDTA, 150 mM KCL, 5 mM MgCl₂, 1.25 g bovine serum

m

L) comprising

6.3.1. 3D-qsar analysis

The molecular modeling studies namely, CoMFA, Advanced

CoMFA and CoMSIA were performed using SYBYL6.9 molecular

m

120

D. Banerjee et al. / European Journal of Medicinal Chemistry 46 (2011) 106e121

modeling software running on a Silicon Graphics Octane R12000³⁸

workstation. The structures were built in 2D using MDL ISIS Draw

2.5 software, and were imported to Discovery Studio 2.0 (Catalyst)

software. CHARMM force ﬁeld was applied to the molecules and

later energy minimized. These 3D structures where then exported

to the SYBYL6.9 workstation where partial charges were calculated

using GasteigereHückel method geometry optimized using Tripos

force ﬁeld with a distance-dependent dielectric function until

a root mean square deviation (RMSD) of 0.01 kcal/mol Å was ach-

ieved. Two different alignment techniques viz. (a) common

substructure ﬁtting method and (b) manual alignment method

were compared to obtain the most efﬁcient alignment for this set of

structurally diverse molecules. The common substructure ﬁtting

method involves superimposition of all the molecules to a common

substructure of the template molecule. In the manual method, the

molecules were aligned manually over a template molecule.

Amongst the two, manual alignment method gave better results.

power of the atomic radii, the electrostatic descriptors are derived

from atomic partial charges, the hydrophobic ﬁelds are derived

from atom-based parameters, and the hydrogen bond donor and

acceptor indices are obtained by a rule-based method derived from

experimental values. In the present work, similarity indices were

computed using a probe atom with radius 1.0 Å, charge þ1,

hydrophobicity þ1, hydrogen bond donor and acceptor properties

of þ1 and attenuation factor of 0.3 for the Gaussian type distance

function [30].

6.3.1.6. Validation of models. PLS analysis was used to correlate the

activities with CoMFA and CoMSIA values containing the steric and

electrostatic potentials. The models were cross-validated using the

Leave One Out (LOO) method by SAMPLS method. The LOO method

involves excluding one compound from the data set and then

deriving a model using the rest. Then with the help of this derived

model, the activity of the excluded molecule is determined. CoMFA

standard scaling was applied to all the CoMFA analyses. The full PLS

analysis was run with a column ﬁltering of 2.0 kcal/mol to reduce

the noise and to speed up the calculations. In case of CoMSIA as

well, SAMPLS method was used. This was followed by a full PLS

analysis, which was run using column ﬁltering of 2.0 kcal/mol.

Autoscaling was applied to all CoMSIA analyses.

6.3.1.1. Data sets. A set of ﬁfty-four molecules, characterized by

a central 2-hydroxy/methoxy carbamothioyl hydrazine carbox-

amide moiety, was divided into a training set and a test set taking

into account either the structural aspects or the anti-HIV inhibitory

proﬁle of compounds. The training set was chosen according to

these criterions: (i) the most- and least-active compounds were

both included.; (ii) compounds were chosen in such a way that the

entire activity range was uniformly sampled; and (iii) structural

superﬂuity was avoided selecting compounds with different

substituents or substitution patterns.

6.3.2. Docking calculations onto HIV-1 RT

A crystallographic structure of HIV-1 RT in complex with

nevirapine (PDB code 1VRT) [31] was used as protein starting

structure for the docking simulation. Each inhibitor was built using

Schrodinger Maestro and energy minimized in the same program

and then docked onto RT using GOLD with the active site being

deﬁned using active atoms generated while redocking nevirapine

onto HIV-1 RT. All the water molecules and the metal ions were

removed from the protein structure prior to docking. Fifty genetic

algorithms were performed for each ligand, with 100 000 energy

evaluations.

6.3.1.2. CoMFA studies. The CoMFA calculations were done using

Tripos Standard and Advanced CoMFA modules in SYBYL. For each

alignment, an sp³carbon atom having a charge of þ1 and a radius of

1.52 Å was used as a probe to calculate various steric and electro-

static ﬁelds, the grid spacing was set to 2 Å and the region was

calculated automatically. To investigate the inﬂuence of different

parameter settings on CoMFA, various steric and electrostatic

cutoffs and grid spacing were set to 30 kcal/mol [29].

6.3.3. Docking calculations onto MTB ICL

Three dimensional coordinates of the MTB ICL/BP complex

(RCSB PDB entry 1F8M) [32] were used as the input structure for

docking simulations performed on Autodock 4.0 due to the highly

rigid structure of the molecule. The cocrystallized ligand was

deleted from the protein structure. The protein was prepared using

Schrodinger’s protein preparation wizard only for hydrogen mini-

mization. Schrodinger Maestro was used for ligand building and

energy minimization and saved in *.mol format and then imported

to Autodock 4.0. Grids of molecular interactions were considered in

a cubic box of 50 ꢃ 50 ꢃ 50 with grid spacing of 0.375 Ǻ. The active

site was centred around the coordinate 19.4465, 41.903, 53.181. Few

of the residues were assigned ﬂexibility. Docking was performed 50

times using the Lamarckian genetic algorithm and a maximum of

250 000 energy evaluations. The ﬁnal docked conformations were

ranked according to their binding free energy.

6.3.1.3. Advanced CoMFA. H-bonding, indicator and parabolic ﬁelds

were used in the Advanced CoMFA routine. Lattice points with steric

energies below the steric cutoff energy were assigned nominal

energies equal to the steric cutoff energies if they were close to H-

bond accepting or donating atoms. Indicator ﬁelds replaced all

lattice energies with magnitudes belowa designated threshold with

zero values. All energies at or above that threshold were replaced

with a nominal energy equal in magnitude to the relevant ﬁeld

cutoff value. When both ﬁelds were included in a single CoMFA

column, the greater of the steric and electrostatic cutoffs was used.

The sign of the original lattice energy is retained. Parabolic ﬁelds are

those in which the magnitude of a standard steric and/or electro-

static ﬁeld at each lattice point has been squared; the original sign of

the energy is retained.

6.3.1.4. CoMFA region focusing. CoMFA region focusing is a method

of application of weights to the lattice points in a CoMFA region to

enhance or attenuate the contribution of these points to subse-

quent analysis. In this study, discriminant power values as weights

and different factors were applied in addition to grid spacing for

getting better models.

Acknowledgment

This work was supported by grants from the Indian Council of

Medical Research, New Delhi, India (58/9/2003-BMS) and Univer-

sity Grant Commission [F.No. 36-61/2008 (SR)], Government of

India.

6.3.1.5. CoMSIA studies. Five similarity ﬁelds namely, steric, elec-

trostatic, and hydrophobic, hydrogen bond donor and acceptor

were calculated. The lattice dimensions were selected with a sufﬁ-

ciently large margin extended up to 4 Å surrounding all aligned

molecules. In CoMSIA, the steric indices are related to the third

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Novel isatinyl thiosemicarbazones derivatives as potential molecule to combat HIV-TB co-infection

DOI: 10.1016/j.ejmech.2010.10.020

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